U.S. patent number 10,946,020 [Application Number 16/091,000] was granted by the patent office on 2021-03-16 for compositions and methods for treating cancer.
This patent grant is currently assigned to University of Virginia Patent Foundation. The grantee listed for this patent is University of Virginia Patent Foundation. Invention is credited to Salome Boroda, Desiree Floyd, Thurl E. Harris, Jr., Inan Olmez, Benjamin Purow.
![](/patent/grant/10946020/US10946020-20210316-C00001.png)
![](/patent/grant/10946020/US10946020-20210316-C00002.png)
![](/patent/grant/10946020/US10946020-20210316-D00001.png)
![](/patent/grant/10946020/US10946020-20210316-D00002.png)
![](/patent/grant/10946020/US10946020-20210316-D00003.png)
![](/patent/grant/10946020/US10946020-20210316-D00004.png)
![](/patent/grant/10946020/US10946020-20210316-D00005.png)
![](/patent/grant/10946020/US10946020-20210316-D00006.png)
![](/patent/grant/10946020/US10946020-20210316-D00007.png)
![](/patent/grant/10946020/US10946020-20210316-D00008.png)
![](/patent/grant/10946020/US10946020-20210316-D00009.png)
View All Diagrams
United States Patent |
10,946,020 |
Purow , et al. |
March 16, 2021 |
Compositions and methods for treating cancer
Abstract
Ritanserin is shown to be an inhibitor of diacylglycerol kinase
alpha (DGK.alpha.) and to be cytotoxic to glioblastoma and melanoma
cells. Ritanserin treatment also provides chemo- and
radiosensitization in cancer cells, prolonged survival in mouse
models of glioblastoma, decreased melanoma tumor burden in mice,
and shows synergistic cytotoxic effects with temozolomide and
chloroquine, both singly and in combination. DGK.alpha. inhibition
with ritanserin shows particular toxicity to the
treatment-resistant mesenchymal subtype in glioblastoma and other
cancers.
Inventors: |
Purow; Benjamin
(Charlottesville, VA), Floyd; Desiree (Gordonsville, VA),
Olmez; Inan (Charlottesville, VA), Harris, Jr.; Thurl E.
(Covesville, VA), Boroda; Salome (Charlottesville, VA) |
Applicant: |
Name |
City |
State |
Country |
Type |
University of Virginia Patent Foundation |
Charlottesville |
VA |
US |
|
|
Assignee: |
University of Virginia Patent
Foundation (Charlottesville, VA)
|
Family
ID: |
1000005422206 |
Appl.
No.: |
16/091,000 |
Filed: |
April 6, 2017 |
PCT
Filed: |
April 06, 2017 |
PCT No.: |
PCT/US2017/026401 |
371(c)(1),(2),(4) Date: |
October 03, 2018 |
PCT
Pub. No.: |
WO2017/177037 |
PCT
Pub. Date: |
October 12, 2017 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20190060317 A1 |
Feb 28, 2019 |
|
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
62319248 |
Apr 6, 2016 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K
45/06 (20130101); A61P 35/00 (20180101); A61K
31/519 (20130101); A61K 31/4706 (20130101); A61K
31/495 (20130101); A61K 31/519 (20130101); A61K
2300/00 (20130101); A61K 31/4706 (20130101); A61K
2300/00 (20130101); A61K 31/495 (20130101); A61K
2300/00 (20130101); A61K 2300/00 (20130101) |
Current International
Class: |
A61K
31/519 (20060101); A61K 45/06 (20060101); A61K
31/495 (20060101); A61K 31/4706 (20060101); A61P
35/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
WO 00/16750 |
|
Mar 2000 |
|
WO |
|
WO 00/57858 |
|
Oct 2000 |
|
WO |
|
WO 2014018563 |
|
Jan 2014 |
|
WO |
|
Other References
Floyd, "ATPS-22 Ritanserin and Temozolomide Synergize Against Human
Glioblastoma In Vitro and in Established Tumors In Vivo", Neuro
Oncol. Nov. 2015; 17(Suppl 5): v22-v23. cited by examiner .
Tso, "Primary Glioblastomas Express Mesenchymal Stem-Like
Properties", Mol Cancer Res 2006;4:607-619. cited by examiner .
Klok et al., "Cholesteryl-(L-Lactic Acid)n Building Blocks for
Self-Assembling Biomaterials," Macromolecules, vol. 35, Iss. 3, pp.
746-759 (2002). cited by applicant .
Lee et al., "The synergistic effect of combination temozolomide and
chloroquine treatment is dependent on autophagy formation and p53
status in glioma cells," Cancer Lett., vol. 360, No. 2, pp. 195-204
(2015). cited by applicant .
Leysen et al., "Receptor-binding properties in vitro and in vivo of
ritanserin: A very potent and long acting serotonin-S2 antagonist,"
Mol Pharmacol., vol. 27, No. 6, pp. 600-611 (1985). cited by
applicant .
Portoghese et al., "Opioid Agonist and Antogonist Bivalent Ligands
as Receptor Probes," Life Sci. vol. 31 pp. 1283-1286 (1982). cited
by applicant .
Portoghese et al., "Opioid Agonist and Antagonist Bivalent Ligands.
The Relationship between Spacer Length and Selectivity at Multiple
Opioid Receptors," J. Med. Chem., vol. 29, No. 10, pp. 1855-1861
(1986). cited by applicant .
Tamiz et al., "Pharmacological and Behavioral Analysis of the
Effects of Some Bivalent Ligand-Based Monoamine Reuptakes
Inhibitors," J. Med. Chem., vol. 44, pp. 1615-1622 (2001). cited by
applicant .
Wittlinger et al., "Time and dose-dependent activation of p53
serine 15 phosphorylation among cell lines with different radiation
sensitivity," Int J Radiat Biol., vol. 83, No. 4, pp. 245-257
(2007). cited by applicant .
Ampie et al., "Heatshock protein vaccines against Glioblastoma:
From bench to bedside," J Neurooncol, vol. 123, No. 3, pp. 441-448
(Jul. 2015). cited by applicant .
Bhat et al., "Mesenchymal Differentiation Mediated by NF-KB
Promotes Radiation Resistance in Glioblastoma," Cancer Cell, vol.
24, No. 3, pp. 331-346 (2013). cited by applicant .
Bullock et al., "Manipulation of Avidity to Improve Effectiveness
of Adoptively Transferred CD8(+) T Cells for Melanoma Immunotherapy
in Human MHC Class I-Transgenic Mice," J Immunol., vol. 167, No.
10, pp. 5824-5831 (2001). cited by applicant .
Chianale et al., "Diacylglycerol Kinase-.alpha. Mediates Hepatocyte
Growth Factor-induced Epithelial Cell Scatter by Regulating Rac
Activation and Membrane Ruffling," Molecular Biology of the Cell,
vol. 18, No. 12, pp. 4859-4871 (Dec. 2007). cited by applicant
.
Chou, "Drug combination studies and their synergy quantification
using the Chou-Talalay method," Cancer Research, vol. 70, No. 2,
pp. 440-446 (2010). cited by applicant .
De Chaffoy de Courcelles et al., "R 59 022, a Diacylglycerol Kinase
Inhibitor: Its effect on Diacylglycerol and Thrombin-induced C
Kinase Activation in the Intact Platelet," J Biol Chem., vol. 60,
No. 29, pp. 15762-15770 (1985). cited by applicant .
Domingo-Musibay et al., "What next for newly diagnosed
glioblastoma?"Future Oncology, vol. 11, No. 24, pp. 3273-3283
(2015). cited by applicant .
Domingues et al., "Diacylglycerol Kinase .alpha. is a Critical
Signalling Node and Novel Therapeutic Target in Glioblastoma and
Other Cancers," Cancer Discovery, vol. 3, No. 7, pp. 782-797 (Jul.
2013). cited by applicant .
Hori et al., "Chloroquine potentiates temozolomide cytotoxicity by
inhibiting mitochondrial autophagy in glioma cells," J Neurooncol.,
vol. 122, No. 1, pp. 11-20 (2015). cited by applicant .
Hussey et al., "Synthesis of a .beta.-Estradiol-Biotin Chimera that
Potently Heterodimerizes Estrogen Receptor and Streptavidin
Proteins in a Yeast Three-Hybrid System," J. Am. Chem. Soc., vol.
125, No. 13, pp. 3692-3693 (2003). cited by applicant .
International Preliminary Report on Patentability corresponding to
International Application No. PCT/US2017/026401 dated Oct. 9, 2018.
cited by applicant .
International Search Report and Written Opinion of the
International Searching Authority corresponding to International
Application No. PCT/US2017/026401 dated Jul. 17, 2017. cited by
applicant .
Kefas et al., "A miR-297/hypoxia/DGK-.alpha. axis regulating
glioblastoma survival," Neuro-Oncology, vol. 15, No. 12, pp.
1652-1663 (2013). cited by applicant .
Kim et al., "Wnt activation is implicated in glioblastoma
radioresistance," Lab Invest., vol. 92, No. 3, pp. 466-473 (2012).
cited by applicant .
Kimura et al., "Chloroquine in Cancer Therapy: A Double-Edged Sword
of Autophagy," Cancer Research, vol. 73, No. 1, pp. 3-7 (2013).
cited by applicant .
Loughery et al., "Critical role for p53-serine 15 phosphorylation
in stimulating transactivation at p53-responsive promoters,"
Nucleic Acids Research, vol. 42, No. 12, pp. 7666-7680 (2014).
cited by applicant .
Mao et al., "Mesenchymal glioma stem cells are maintained by
activated glycolytic metabolism involving aldehyde dehydrogenase
1A3," Proc Natl Acad Sci, vol. 110, No. 21, pp. 8644-8649 (2013).
cited by applicant .
Olenchock et al., "Disruption of diacylglycerol metabolism impairs
the induction of T cell anergy," Nat Immunol., vol. 7, No. 11, pp.
1174-1181 (2006). cited by applicant .
Olin et al., "Superior Efficacy of Tumor Cell Vaccines Grown in
Physiologic Oxygen," Clinical Cancer Res., vol. 16, No. 19, pp.
4800-4808 (2010). cited by applicant .
Ostrom et al., "CBTRUS Statistical Report: Primary Brain and
Central Nervous System Tumors Diagnosed in the United States in
2008-2012," Neuro-Oncol., vol. 17, Suppl 4, pp. iv1-iv62 (2015).
cited by applicant .
Paar et al., "Bivalent Ligands with Rigid Double-Stranded DNA
Spacers Reveal Structural Constraints on Signaling by Fc Rl1," J.
Immunol., vol. 169, pp. 856-864 (2002). cited by applicant .
Phillips et al., "Molecular subclasses of high-grade glioma predict
prognosis, delineate a pattern of disease progression, and resemble
stages in neurogenesis," Cancer Cell, vol. 9, No. 3, pp. 157-173
(2006). cited by applicant .
Purow, "Molecular Pathways: Targeting Diacylglycerol Kinase Alpha
in Cancer," Clinical Cancer Research, vol. 21, No. 22, pp.
5008-5012 (Nov. 2015). cited by applicant .
Roller et al., "Synthetic Lethal Screening with Small-Molecule
Inhibitors Provides a Pathway to Rational Combination Therapies for
Melanoma," Mol Cancer Ther., vol. 11, No. 11, pp. 2505-2515 (2012).
cited by applicant .
Verhaak et al., "Integrated Genomic Analysis Identifies Clinically
Relevant Subtypes of Glioblastoma Characterized by Abnormalities in
PDGFRA, IDH1, EGFR, and NF1," Cancer Cell, vol. 17, No. 1, pp.
98-110 (2010). cited by applicant .
Zha et al., "T cell anergy is reversed by active Ras and is
regulated by diacylglycerol kinase-.alpha.," Nat Immunol., vol. 7,
No. 11, pp. 1166-1173 (2006). cited by applicant .
Nobe et al., "Effect of diacylglycerol kinase inhibitor, R59022 on
cytosolic free calcium level and force development in guinea pig
taenia coli.," Res Commun Chem Pathol Pharmacol., vol. 81, No. 3,
pp. 331-343 (1993). [abstract only]. cited by applicant .
Olmez et al., "Targeting the mesenchymal subtype in glioblastoma
and other cancers via inhibition of diacylglycerol kinase alpha,"
Neuro-Oncology, pp. 1-11 (2017). cited by applicant .
Sato et al., "Evaluations of the Selectivities of the
Diacylglycerol Kinase Inhibitors R59022 and R59949 Among
Diacylglycerol Kinase Isozymes Using a New Non-Radioactive Assay
Method," Pharmacology, vol. 92, No. 1-2, pp. 99-107 (2013). cited
by applicant .
Stepinski et al., "Use of hydrophilic diamines for bridging of two
opioid peptide pharmacophores," Internat. J. of Peptide &
Protein Res. vol. 38, pp. 588-592 (1991). cited by applicant .
Timmerman et al., "Deuterated ritanserin analysis by gas
chromatography/mass spectrometry: a sensitive technique to study
human ritanserin pharmacokinetics," Biomed Environ Mass Spectrom,
vol. 18, No. 7, pp. 498-502 (1989). cited by applicant.
|
Primary Examiner: Javanmard; Sahar
Attorney, Agent or Firm: Jenkins, Wilson, Taylor & Hunt,
P.A.
Government Interests
GRANT STATEMENT
This invention was made with government support under Grant Nos. CA
180699 and CA 189524 awarded by the National Institutes of Health.
The government has certain rights in the invention.
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a national phase application of PCT
International Patent Application PCT/US2017/026401, filed Apr. 6,
2017, which claims the benefit of U.S. Provisional Patent
Application Ser. No. 62/319,248, filed Apr. 6, 2016, each of which
is incorporated herein by reference in its entirety.
Claims
What is claimed is:
1. A method for treating a cancer in a subject in need thereof,
wherein the method comprises administering to the subject a
therapeutically effective amount of ritanserin, wherein the cancer
is selected from the group consisting of mesenchymal cancer,
melanoma and glioblastoma.
2. The method of claim 1, wherein the method further comprises
administering at least one second treatment to the subject.
3. The method of claim 2, wherein the at least one second treatment
is selected from the group consisting of a surgical resection of a
tumor, radiotherapy, immunotherapy, alternating electric field
therapy, and chemotherapy.
4. The method of claim 2, wherein the method comprises
administering to the subject at least one second treatment selected
from the group consisting of temozolomide (TMZ), chloroquine,
bevacizumab, imatinib, radiation, and an immunotherapeutic
agent.
5. The method of claim 2, wherein the method comprises
administering to the subject at least two second treatments,
wherein the at least two second treatments have synergistic
activity with each other.
6. The method of claim 5, wherein the at least two second
treatments are TMZ and chloroquine.
7. The method of claim 1, wherein the ritanserin is administered
orally to the subject.
8. The method of claim 1, wherein the subject is human.
9. A method of inducing chemo- or radiosensitivity in a subject
undergoing or scheduled to undergo treatment with a
chemotherapeutic agent or radiation to treat a disease or disorder
treatable thereby, the method comprising administering ritanserin
to the subject, wherein the disease or disorder is selected from
the group consisting of mesenchymal cancer.
10. The method of claim 9, wherein administering the ritanserin is
performed prior to and/or concurrently with the administration of a
chemotherapeutic agent.
11. The method of claim 9, wherein administering the ritanserin is
performed prior to and/or concurrently with the administration of
radiation.
12. A method for inhibiting a cancer in a subject in need thereof,
wherein the method comprises administering to the subject a
therapeutically effective amount of ritanserin, wherein the cancer
is a melanoma or a glioblastoma.
Description
TECHNICAL FIELD
The presently disclosed subject matter provides compositions
comprising ritanserin or analog thereof for treating cancers, such
as glioblastomas and melanomas, and for inhibiting diacylglycerol
kinase alpha. Also provided are methods of treating cancer using
ritanserin or analog thereof, alone or in combination with other
agents.
ABBREVIATIONS
.degree. C.=degrees Celsius %=percentage .mu.g=microgram
.mu.l=microliter .mu.M=micromolar CI=combination index
CQ=chloroquine DAG=diacylglycerol DGK.alpha.=Diacylglycerol Kinase
alpha DMSO=dimethylsulfoxide em.=emission g=grams GBM=glioblastoma
multiforme IC.sub.50=50% inhibitory concentration kg=kilogram
min=minute mg=milligram ml=milliliter mm=millimeter mM=millimolar
MRI=magnetic resonance imaging N=normal nm=nanometer
PA=phosphatidic acid RFU=relative fluorescence units
TMZ=temozolomide v=volume
BACKGROUND
Glioblastoma multiforme (GBM) is an incurable Grade 4 tumor of the
central nervous system, comprising nearly half of malignant adult
primary brain tumors. See Ostrom et al., 2015. Recent clinical
trials with electric fields have improved average survival times
when added to the standard regimen of surgery, radiation, and
temozolomide (TMZ), but median survival is still under two years.
See Domingo-Musibay et al., 2015.
Diacylglycerol Kinase alpha (DGK.alpha.), a highly conserved enzyme
that catalyzes the conversion of diacylglycerol (DAG) to
phosphatidic acid (PA), has recently been shown to be a cancer
therapeutic target upstream of mTOR, HIF-1.alpha., and cAMP
signaling. See Dominguez et al., 2013; and Kefas et al., 2013. More
particularly, GBM and melanoma cells have been found to be
sensitive to DGK.alpha. knockdown and to inhibition with the
DGK.alpha. inhibitors R59022 and R59949. See Dominguez et al.,
2013. DGK.alpha. inhibition has also been shown to prevent T cell
anergy, indicating that it can be a cancer immunotherapy target.
See Olenchock et al., 2006; and Zha et al., 2006.
R59022 was first described several decades ago (see de Chaffoy de
Dourcelles et al., 1985.), but only recently shown to be cytotoxic
to cancer. R59022 has not been studied in humans, and animal models
have been mostly ex vivo. See Nobe et al., 1993. In recent mouse
models, R59022 delayed subcutaneous melanoma progression, but had
limited efficacy in orthotopic glioblastoma, perhaps due to limited
blood-brain barrier penetrance and rapid clearance from the serum.
See Dominguez et al., 2013.
Accordingly, there is an ongoing need for additional compositions
and methods useful for treating diseases and disorders involving
DGK.alpha. and its signaling pathways, such as cancer. In
particular, there is an ongoing need for additional
pharmaceutically active agents and methods for treating GBM and
other cancers. For example, there is a need for additional agents
that can inhibit DGK.alpha. and that can cross the blood-brain
barrier. There is also a need for DGK.alpha. inhibitors that can
increase the efficacy of other treatments for GBM and other
cancers.
SUMMARY
In some embodiments, the presently disclosed subject matter
provides a method for treating a cancer in a subject in need
thereof, wherein the method comprises administering to the subject
a therapeutically effective amount of ritanserin or analog thereof.
In some embodiments, the cancer is a melanoma or a glioblastoma. In
some embodiments, the cancer is a mesenchymal cancer. In some
embodiments, the cancer is a mesenchymal glioblastoma.
In some embodiments, the method further comprises administering at
least one second treatment to the subject. In some embodiments, the
at least one second treatment is selected from the group comprising
a surgical resection of a tumor, radiotherapy, immunotherapy,
alternating electric field therapy, or chemotherapy. In some
embodiments, the method comprises administering to the subject at
least one second treatment selected from the group comprising
temozolomide (TMZ), chloroquine, bevacizumab, imatinib, radiation,
or an immunotherapeutic agent.
In some embodiments, the method comprises administering to the
subject at least two second treatments, wherein the at least two
second treatments have synergistic activity with each other. In
some embodiments, the at least two second treatments are TMZ and
chloroquine.
In some embodiments, the ritanserin is administered orally to the
subject. In some embodiments, the subject is human.
In some embodiments, the presently disclosed subject matter
provides a composition comprising ritanserin, or analog thereof;
and at least one of temozolomide (TMZ), chloroquine, bevacizumab,
imatinib, or an immunotherapeutic agent. In some embodiments, the
composition comprises ritanserin, TMZ, and chloroquine.
In some embodiments, the composition further comprises a
pharmaceutically acceptable excipient. In some embodiments, the
pharmaceutically acceptable excipient is selected from the group
comprising granulating agents, binding agents, lubricating agents,
disintegrating agents, sweetening agents, glidants, anti-adherents,
anti-static agents, surfactants, anti-oxidants, gums, coloring
agents, flavouring agents, coating agents, plasticizers,
preservatives, suspending agents, emulsifying agents,
spheronization agents or combinations thereof. In some embodiments,
the composition is formulated for oral administration.
In some embodiments, the presently disclosed subject matter
provides a method of inhibiting diacylglycerol kinase alpha
(DGK.alpha.) in a biological sample, wherein the method comprises
contacting the sample with ritanserin or analog thereof. In some
embodiments, the biological sample is selected from the group
comprising a cell, a tissue, an organ or a subject.
In some embodiments, the presently disclosed subject matter
provides a method of inducing chemo- or radiosensitivity in a
subject undergoing or scheduled to undergo treatment with a
chemotherapeutic agent or radiation to treat a disease or disorder
treatable thereby, the method comprising administering ritanserin
or analog thereof to the subject. In some embodiments,
administering the ritanserin is performed prior to and/or
concurrently with the administration of a chemotherapeutic agent.
In some embodiments, administering the ritanserin is performed
prior to and/or concurrently with the administration of
radiation.
In some embodiments, the disease or disorder is a cancer. In some
embodiments, the cancer is a mesenchymal cancer. In some
embodiments, the cancer is a melanoma or a glioblastoma.
Accordingly, it is an object of the presently disclosed subject
matter to provide compositions and methods for treating cancer
and/or inhibiting DGK.alpha..
An object of the presently disclosed subject matter having been
stated hereinabove, and which is achieved in whole or in part by
the presently disclosed subject matter, other objects will become
evident as the description proceeds hereinbelow.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A is a schematic drawing of the chemical structures of
R59949, R59022, and ritanserin.
FIG. 1B is a graph showing the drug dose-dependent Diacylglycerol
Kinase alpha (DGK.alpha.) activity curve with R59022. The 50
percent inhibitory concentration (IC.sub.50) is 20 micromolar
(.mu.M), as shown by a logarithmic scale with the highest
concentration of drug being 100 .mu.M.
FIG. 1C is a graph showing the drug dose-dependent Diacylglycerol
Kinase alpha (DGK.alpha.) activity curve with ritanserin. The 50
percent inhibitory concentration (IC.sub.50) is 9.0 micromolar
(.mu.M), as shown by a logarithmic scale with the highest
concentration of drug being 100 .mu.M.
FIG. 1D is a graph showing the conversion of diacylglycerol (DAG)
to phosphatidic acid (PA) by purified Diacylglycerol Kinase alpha
(DGK.alpha.) as measured by mass spectrometry with increasing
adenosine triphosphate (ATP) concentration, in the presence of two
concentrations of R59022 (0.5 mole (mol) % R59022, squares; or 2.0
mol % R59022, upward-pointing triangles) or ritanserin (0.5 mol %
Ritanserin, downward-pointing triangles; or 2.0 mol % Ritanserin,
diamonds) or in the absence of drug (NO DRUG, circles).
FIG. 2A is a series of graphs showing the cytotoxic effect of
ritanserin on different cancer cell lines: U251 human glioblastoma
cells (upper left), VMM39 human melanoma cells (upper right), GL261
murine glioma cells (lower left) and JWL578 human glioblastoma stem
cells (lower right). The number of live cells was assessed four
days post-treatment with ritanserin at concentrations between 0
micromolar (.mu.M) and 17.5 or 20 .mu.M. Error bars represent
standard deviation. P values shown were generated from one-way
analysis of variance (ANOVA) with Tukey's post-test for multiple
comparisons (p<0.05, *, p<0.01, **, p<0.001, ***,
p<0.0001, ****).
FIG. 2B is a series of plots of Annexin V positive U251 human
glioblastoma cells after treatment with vehicle (dimethylsulfoxide,
DMSO) (left), with 15 micromolar (.mu.M) ritanserin (middle), or
with 20 .mu.M ritanserin (right). The plots represent gating on
live cells and 10,000 events were counted in three replicates per
treatment.
FIG. 2C is (left) a plot of relative fluorescence units (RFU) of
U251 human glioblastoma cells treated with 0 to 22.5 micromolar
(.mu.M) ritanserin alone (circles) or a combination of 0 to 22.5
.mu.M ritanserin and 50 .mu.M chloroquine (CQ) (squares). Cells
were treated for three days and an alamarBlue assays was performed.
Error bars represent standard deviation. P values shown were
generated from one-way analysis of variance (ANOVA) with Tukey's
post-test for multiple comparisons (p<0.05, *, p<0.01, **,
p<0.001, ***, p<0.0001, ****). In the upper right is a
radiograph of an immunoblot of lysates of ritanserin-treated U251
cells. The lysates were made after 24 hours of treatment and
immunoblotted for autophagy indicator LC3 A/B II. In the bottom
right is a combination index generated by the Chou-Talalay method
for determining synergy based on data for the combination of
ritanserin and CQ.
FIG. 2D is a radiograph of an immunoblot of U251 human glioblastoma
cells treated only with vehicle (dimethylsulfoxide, DMSO) or with
15 .mu.M ritanserin and assayed for poly ADP Ribose Polymerase
(PARP) cleavage.
FIG. 2E is a graph showing the cytotoxic effects of ritanserin on
murine B16 melanoma cells. The number of live cells was assessed
four days post-treatment with ritanserin at concentrations between
0 micromolar (.mu.M) and 20 .mu.M. Error bars represent standard
deviation. P values shown were generated from one-way analysis of
variance (ANOVA) with Tukey's post-test for multiple comparisons
(p<0.05, *, p<0.01, **, p<0.001, ***, p<0.0001,
****).
FIG. 2F is a series of plots of Annexin V positive VMM39 human
melanoma cells after treatment with 10 micromolar (.mu.M)
ritanserin (second from left), 12.5 .mu.M ritanserin (center), 15
.mu.M ritanserin (second from right) or 20 .mu.M ritanserin
(right). For comparison, a plot of positive cells from a control
sample treated only with vehicle (dimethylsulfoxide, DMSO) is also
shown (left). The plots represent gating on live cells and 10,000
events were counted in three replicates per treatment.
FIG. 2G is a series of graphs showing the cytotoxic effects of
ritanserin (0 to 22.5 micromolar (.mu.M) ritanserin) on
necrostatin-1 pretreated U251 human glioblastoma cells (left),
necrostatin-1 pretreated VMM39 human melanoma cells (center) and on
ZVAD-FMK pre-treated U251 human glioblastoma cells (right). Cell
survival data is provided as relative fluorescence units (RFU)
measured with alamarBlue reagent. Pretreatment with necrostatin-1
was performed with 50 .mu.M necrostatin-1. Pretreatment with
ZVAD-FMK was performed with 50 .mu.M ZVAD-FMK. Error bars represent
standard deviation. P values shown were generated from one-way
analysis of variance (ANOVA) with Tukey's post-test for multiple
comparisons (p<0.05, *, p<0.01, **, p<0.001, ***,
p<0.0001, ****).
FIG. 2H are radiographs of immunoblots of lysates of U251 cells
treated with 20 micromolar (.mu.M) chloroquine (CQ), 10 .mu.M
ritanserin (Rit), or a combination of 20 .mu.M CQ and 10 .mu.M
ritanserin. The lysates were made after 48 hours of treatment and
immunoblotted for autophagy indicator LC3 A/B II and p62.
FIG. 3A is a pair of graphs showing the effects of phosphatidic
acid (PA) on ritanserin cytoxicity in U251 human glioblastoma cells
(left) and JWL578 human glioblastoma stem cells (right). U251 and
JWL cells were incubated with 10 micromolar (.mu.M) ritanserin or
dimethylsulfoxide (DMSO) and either vehicle (2:1
chloroform:methanol; volume:volume; black bars) or 100 .mu.M PA
(grey bars). Live cells were collected and counted 48 hours later.
Student's T-test or one-way analysis of variance (ANOVA) with
Tukey's post-test was used for analysis (p<0.01, **, p<0.001,
***).
FIG. 3B is a series of graphs showing mechanistic target of
rapamycin (mTOR) promoter reporter activity in U251 human
glioblastoma cells (left) and VMM39 human melanoma cells (center)
or hypoxia-inducible factor 1 alpha (HIF-1.alpha.) binding activity
in U251 human glioblastoma cells (right) measured in response to 20
micromolar (.mu.M) ritanserin (grey bars) or dimethylsulfoxide
(DMSO) in a dual luciferase assay. Student's T-test or one-way
analysis of variance (ANOVA) with Tukey's post-test was used for
analysis (p<0.01, **, p<0.001, ***).
FIG. 3C a pair of graphs showing the effects of phosphatidic acid
(PA) on ritanserin cytoxicity in U251 human glioblastoma cells
(left) and JWL578 human glioblastoma stem cells (right). U251 and
JWL cells were incubated with 10 micromolar (.mu.M) ritanserin or
dimethylsulfoxide (DMSO) and either vehicle (media, black bars) or
100 .mu.M PA (grey bars). Live cells were collected and counted 48
hours later. Student's T-test or one-way analysis of variance
(ANOVA) with Tukey's post-test was used for analysis (p<0.1, *,
p<0.01, **, p<0.001, ***).
FIG. 4A is a series of graphs of expression of HSP60 (left), p53
S15 (center) or p70 S6K T389 (right) in U251 human glioblastoma
cells treated with 15 micromolar (.mu.M) ritanserin (grey bars) or
dimethylsulfoxide (DMSO) (black bars). Cells were treated with
ritanserin or DMSO and 24 hour lysates were used in a human
phospho-proteomics assay. Data represents corrected average spot
densities. Student's T-test was used for analysis, 2 replicates per
group (p<0.01, **, p<0.001, ***, p<0.0001, ****).
FIG. 4B is a group of radiographs of immunoblots confirming the
protein targets for the data described for FIG. 4A using U251 human
glioblastoma cells (left) and VMM39 human melanoma cells
(right).
FIG. 5A is a graph showing the survival curves of athymic nu/nu
mice implanted intracranially with U251 human glioblastoma cells
and treated with 10 milligrams per kilogram (mg/kg) ritanserin
(squares) or 50 mg/kg ritanserin (triangles). For comparison, the
survival curve of mice implanted with U251 human glioblastoma cells
and treated with vehicle (corn oil) is also shown (circles). Daily
treatment with ritanserin or corn oil was started seven days after
implantation of the glioblastoma cells. Seven mice were used per
group. Kaplan-Meier analysis was used for survival curve generation
(p<0.01,**).
FIG. 5B is (top) a graph showing the survival curves of wild type
C57BL/6 mice implanted intracranially with GL261 murine glioma
cells and treated daily after six days with 50 milligrams per
kilogram (mg/kg) ritanserin (triangles) or vehicle (corn oil)
(circles); and (bottom) magnetic resonance imaging (MRI) images of
the mice performed three weeks after implantation of the glioma
cells. Seven mice were used per group. Kaplan-Meier analysis was
used for survival curve generation (p<0.01,**).
FIG. 5C is a graph showing tumor volume in athymic nu/nu mice
implanted subcutaneously with VMM39 human melanoma cells and
treated daily with 10 milligrams per kilogram (mg/kg) ritanserin
(squares), 50 mg/kg ritanserin (triangles) or vehicle (corn oil,
circles) after one week. Tumor volume was assayed using electronic
calipers three weeks post implantation. Significance was calculated
using Student's t-test (p<0.001, ***).
FIG. 5D is (left) a graph showing the survival curves of athymic
nu/nu mice implanted intracranially with A375 human melanoma cells
and treated with 50 milligrams per kilogram (mg/kg) ritanserin
(triangles) or vehicle (corn oil) (circles) daily starting six days
after implantation; (center) a graph showing the average tumor
volumes three weeks post implantation in the same mice (vehicle
treated mouse data shown in black bars and ritanserin treated mouse
data shown in grey bars); and (right) the magnetic resonance
imaging images of the mice performed three weeks post
implantation.
FIG. 5E is a graph showing tumor volumes of wild type C57BL/6 mice
implanted intracranially with GL261 murine glioma cells and treated
daily after six days with 100 milligrams per kilogram (mg/kg)
ritanserin (grey bars) or vehicle (corn oil) (black bars). Magnetic
resonance imaging (MRI) images of the mice are shown to the right
of the graph.
FIG. 6A is a graph of cell survival (as measured by relative
fluorescence units (RFU)) in U251 human glioblastoma cells treated
with a combination 0 to 17.5 micromolar (.mu.M) ritanserin and 0
.mu.M temozolomide (TMZ) (circles), 25 .mu.M TMZ (squares), 50
.mu.M TMZ (upward-pointing triangles) or 75 .mu.M TMZ
(downward-pointing triangles). Fluorescence data was taken from an
alamarBlue assay performed after 4 days of incubation of the cells
with the ritanserin and TMZ combinations.
FIG. 6B is a graph of cell survival (as measured by relative
fluorescence units (RFU)) in VMM39 human melanoma cells treated
with a combination 0 to 22.5 micromolar (.mu.M) ritanserin and 0
.mu.M temozolomide (TMZ) (circles), 25 .mu.M TMZ (squares), 50
.mu.M TMZ (upward-pointing triangles), 75 .mu.M TMZ
(downward-pointing triangles), 100 .mu.M TMZ (diamonds) or 150
.mu.M TMZ (open squares). Fluorescence data was taken from an
alamarBlue assay performed after 4 days of incubation of the cells
with the ritanserin and TMZ combinations.
FIG. 6C is a pair of combination index plots prepared using the
Chou-Talalay method for determining synergy from the data described
for FIG. 6A (left) and FIG. 6B (right).
FIG. 6D is (left) a graph of cell survival (as measured by relative
fluorescence units (RFU)) in U251 human melanoma cells treated with
0 to 25 micromolar (.mu.M) ritanserin and a mixture of 20 .mu.M
chloroquine (CQ) and 75 .mu.M temozolomide (TMZ) (squares), 75
.mu.M TMZ (diamonds), 20 .mu.M CQ (triangles) or ritanserin alone
(i.e., 0 .mu.M CQ and 0 .mu.M TMZ) (circles); and (right) a
combination index plot of synergy for the combination of
ritanserin, CQ and TMZ prepared using the Chou-Talalay method.
Fluorescence data was collected using alamarBlue assay after 4 days
of treatment.
FIG. 6E is a graph of cell survival (as measured by relative
fluorescence units (RFU)) in G2 mesenchymal glioblastoma stem cells
treated with 0 to 25 micromolar (.mu.M) ritanserin and 0 .mu.M
temozolomide (TMZ) (circles), 25 .mu.M TMZ (squares), 50 .mu.M TMZ
(upward-pointing triangles), or 100 .mu.M TMZ (downward-pointing
triangles). Fluorescence data was taken from an alamarBlue assay
performed after 4 days of incubation of the cells with the
ritanserin and TMZ combinations.
FIG. 7A is a graph showing the synergistic cytotoxic effects of
chloroquine (CQ) and ritanserin (Rit) on U251 human glioblastoma
cells. Cells were treated with 20 micromolar (.mu.M) CQ and/or 10
.mu.M Rit and then cell survival was analyzed 72-96 hours later by
cell counting. For comparison, data (provided in cells per well) is
also provided for cells treated with vehicle.
FIG. 7B is a graph showing the synergistic cytotoxic effects of
chloroquine (CQ) and ritanserin (Rit) on GL261 mouse glioma cells.
Cells were treated with 10 micromolar (.mu.M) CQ and/or 10 .mu.M
Rit and then cell survival was analyzed 72-96 hours later via
alamarBlue assay. For comparison, data (provided in relative
fluorescence units (RFU)) is also provided for cells treated with
vehicle.
FIG. 7C is a graph showing the synergistic cytotoxic effects of
chloroquine (CQ) and ritanserin (Rit) on GL261 mouse glioma cells.
Cells were treated with 10 micromolar (.mu.M) CQ and/or 10 .mu.M
Rit and then cell survival was analyzed 72-96 hours later by cell
counting. For comparison, data (provided in cells per well) is also
provided for cells treated with vehicle.
FIG. 7D is a graph showing the effects of chloroquine (CQ) and
ritanserin (Rit) on immortalized astrocytes. Cells were treated
with 10 micromolar (.mu.M) CQ and/or 10 .mu.M or 15 .mu.M Rit and
then cell survival was analyzed 72-96 hours later via alamarBlue
assay. For comparison, data (provided in relative fluorescence
units (RFU)) is also provided for cells treated with vehicle.
FIG. 7E is a graph showing the synergistic cytotoxic effects of
chloroquine (CQ) and ritanserin (Rit) on VMM49 human melanoma
cells. Cells were treated with 10 micromolar (.mu.M) CQ and/or 15
.mu.M Rit and then cell survival was analyzed 72-96 hours later via
alamarBlue assay. For comparison, data (provided in relative
fluorescence units (RFU)) is also provided for cells treated with
vehicle.
FIG. 7F is a graph showing the synergistic cytotoxic effects of
chloroquine (CQ) and ritanserin (Rit) on B16 mouse melanoma cells.
Cells were treated with 5 micromolar (.mu.M) CQ and/or 10 .mu.M Rit
and then cell survival was analyzed 72-96 hours later via
alamarBlue assay. For comparison, data (provided in relative
fluorescence units (RFU)) is also provided for cells treated with
vehicle.
FIG. 7G is a graph showing the synergistic cytotoxic effects of
chloroquine (CQ) and ritanserin (Rit) on G528 human glioblastoma
stem cells. Cells were treated with 10 micromolar (.mu.M) CQ and/or
10 .mu.M or 15 .mu.M Rit and then cell survival was analyzed 72-96
hours later via alamarBlue assay. For comparison, data (provided in
relative fluorescence units (RFU)) is also provided for cells
treated with vehicle.
FIG. 7H is a graph showing the synergistic cytotoxic effects of
chloroquine (CQ) and ritanserin (Rit) on G34 human glioblastoma
stem cells. Cells were treated with 5 micromolar (.mu.M) CQ and/or
10 .mu.M and then cell survival was analyzed 72-96 hours later via
alamarBlue assay. For comparison, data (provided in relative
fluorescence units (RFU)) is also provided for cells treated with
vehicle.
FIG. 8A is a graph showing the effects of treatment with varying
concentrations of ritanserin on 5 different mesenchymal
glioblastoma cell lines (81, 62, 2, 88, and 34) and 6
non-mesenchymal glioblastoma cell lines (464, 816, 44, 559, 528,
and 827).
FIG. 8B is a graph showing the effects of ritanserin (checked bars)
on two different proneural subtype glioblastoma cell lines (816 and
464) and on the same cell lines following TNF alpha induced
proneural-to-mesenchymal transition (816+THF and 464+TNF). Data for
cells treated with vehicle (control) are shown in the black
bars.
FIG. 8C is a graph of survival curves of mice implanted
intracranially with G88 mesenchymal glioblastoma stem cells and
treated with 50 milligrams per kilogram (mg/kg) ritanserin or
vehicle (corn oil) daily starting six days after implantation.
FIG. 8D is a graph showing that lung and pancreatic carcinoma cells
become more sensitive to ritanserin treatment (15 micromolar
(.mu.M) and 10 .mu.M, respectively) after epithelial-mesenchymal
transition (EMT) induced with TNF-alpha and TGF-beta. "Parental
Ctrl" indicates data for cells prior to EMT and without ritanserin
treatment; "Parental Rit" indicates data for cells prior to EMT and
with ritanserin treatment; "EMT Ctrl" indicates data for cells
after EMT and without ritanserin treatment; and "EMT Rit" indicates
data for cells after EMT and with ritanserin treatment. Two-way
analysis of variance (ANOVA) with Bonferroni correction was used
for analysis (**P<0.01, ***P<0.0001). All values are
mean.+-.SEM of triplicates.
FIG. 8E is a graph showing that small interfering ribonucleic acid
(siRNA) for inhibiting diacylglycerol kinase alpha (DGK.alpha.)
exhibits greater cytotoxicity in mesenchymal (MES) glioblastoma
cell lines (GSC) versus non-mesenchymal lines (e.g., proneural (PN)
or classical (CL) lines). One-way analysis of variance (ANOVA) with
post-hoc Tukey was used for analysis (***P<0.0001).
FIG. 8F is a graph of the survival curves of mice implanted
intercranially with G2 mesenchymal (MES) glioblastoma stem cells
and treated with 50 milligrams per kilogram (mg/kg) ritanserin or
vehicle (corn oil) via oral gavage daily starting six days after
implantation.
FIG. 9A is a graph showing the effects of radiation and ritanserin
treatment on G2 mesenchymal glioblastoma stem cells. The stem cells
were treated with 1.25 micromolar (.mu.M) ritanserin or an equal
volume:volume amount of vehicle and then treated with or without 4
gray (Gy) radiation prior to plating in soft agar colonies. After
two weeks, colony numbers were counted. For comparison, data is
also provided for untreated control cells.
FIG. 9B is a graph showing the effects of radiation and ritanserin
treatment on radioresistant G88 mesenchymal glioblastoma stem
cells. The stem cells were treated with 1.25 micromolar (.mu.M)
ritanserin or an equal volume:volume amount of vehicle and then
treated with or without 4 gray (Gy) radiation prior to plating in
soft agar colonies. After two weeks, colony numbers were counted.
For comparison, data is also provided for untreated control
cells.
FIG. 9C are autoradiographs from an immunoblot assay of cells
treated with ritanserin, radiation, or a combination of ritanserin
and radiation, showing the effects of ritanserin on downstream
effectors of radiation response, i.e., p-ATM, p-Chk1 s345, p-Chk2
T68, and p-p53 s15.
FIG. 9D is a graph showing the effect of ritanserin treatment on
NF-.kappa.B expression in G2 mesenchymal glioblastoma cells.
NF-.kappa.B expression was measured using a luciferase assay.
FIG. 9E is a graph showing the effect of ritanserin treatment on
NF-.kappa.B expression in G88 mesenchymal glioblastoma cells.
NF-.kappa.B expression was measured using a luciferase assay.
FIG. 10A is a graph showing ritanserin inhibition of RhoA measured
via an enzyme-linked immunosorbent assay (ELISA) for RhoA in G2
mesenchymal glioblastoma and G88 mesenchymal glioblastoma
cells.
FIG. 10B is a radiograph of the effects of ritanserin on levels of
unprenylated ras-related protein Rap1A in G2 and G88 mesenchymal
glioblastoma cells and in 816 non-mesenchymal glioblastoma
cells.
FIG. 11A is a radiograph of an immunblot of three mesenchymal
glioblastoma stem cell lines showing the activation of
platelet-derived growth factor receptor alpha (PDGFR.alpha.)
following three days of 3 micromolar (3 .mu.M) ritanserin
treatment.
FIG. 11B is a graph showing the cytotoxic synergy between
ritanserin (2 micromolar (.mu.M)) and imatinib (0.75 .mu.M) in G2
and G88 mesenchymal glioblastoma stem cells following five days of
treatment. Significance was calculated using One-way analysis of
variance (ANOVA) with post-hoc Tukey analysis (**P<0.005). All
values are mean.+-.SEM of triplicates.
DETAILED DESCRIPTION
The presently disclosed subject matter will now be described more
fully. The presently disclosed subject matter can, however, be
embodied in different forms and should not be construed as limited
to the embodiments set forth herein below and in the accompanying
Examples. Rather, these embodiments are provided so that this
disclosure will be thorough and complete, and will fully convey the
scope of the embodiments to those skilled in the art.
All references listed herein, including but not limited to all
patents, patent applications and publications thereof, and
scientific journal articles, are incorporated herein by reference
in their entireties to the extent that they supplement, explain,
provide a background for, or teach methodology, techniques, and/or
compositions employed herein.
I. Definitions
While the following terms are believed to be well understood by one
of ordinary skill in the art, the following definitions are set
forth to facilitate explanation of the presently disclosed subject
matter.
Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood to one of
ordinary skill in the art to which the presently disclosed subject
matter belongs.
Following long-standing patent law convention, the terms "a", "an",
and "the" refer to "one or more" when used in this application,
including the claims.
The term "and/or" when used in describing two or more items or
conditions, refers to situations where all named items or
conditions are present or applicable, or to situations wherein only
one (or less than all) of the items or conditions is present or
applicable.
The use of the term "or" in the claims is used to mean "and/or"
unless explicitly indicated to refer to alternatives only or the
alternatives are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and
"and/or." As used herein "another" can mean at least a second or
more.
The term "comprising," which is synonymous with "including,"
"containing," or "characterized by," is inclusive or open-ended and
does not exclude additional, unrecited elements or method steps.
"Comprising" is a term of art used in claim language which means
that the named elements are essential, but other elements can be
added and still form a construct within the scope of the claim.
As used herein, the phrase "consisting of" excludes any element,
step, or ingredient not specified in the claim. When the phrase
"consists of" appears in a clause of the body of a claim, rather
than immediately following the preamble, it limits only the element
set forth in that clause; other elements are not excluded from the
claim as a whole.
As used herein, the phrase "consisting essentially of" limits the
scope of a claim to the specified materials or steps, plus those
that do not materially affect the basic and novel characteristic(s)
of the claimed subject matter.
With respect to the terms "comprising," "consisting of," and
"consisting essentially of," where one of these three terms is used
herein, the presently disclosed and claimed subject matter can
include the use of either of the other two terms.
Unless otherwise indicated, all numbers expressing quantities of
temperature, time, concentration, and so forth used in the
specification and claims are to be understood as being modified in
all instances by the term "about." Accordingly, unless indicated to
the contrary, the numerical parameters set forth in this
specification and attached claims are approximations that can vary
depending upon the desired properties sought to be obtained by the
presently disclosed subject matter.
As used herein, the term "about," when referring to a value is
meant to encompass variations of in one example .+-.20% or .+-.10%,
in another example .+-.5%, in another example .+-.1%, and in still
another example .+-.0.1% from the specified amount, as such
variations are appropriate to perform the disclosed methods.
Numerical ranges recited herein by endpoints include all numbers
and fractions subsumed within that range (e.g. 1 to 5 includes, but
is not limited to, 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5).
A "compound," as used herein, refers to any type of chemical or
biochemical substance or agent that is commonly considered a drug,
or a candidate for use as a drug, as well as combinations and
mixtures of the above.
The term "active compound" as used herein refers to ritanserin and
derivatives or analogs thereof, particularly those that have the
ability to inhibit DGK.alpha. and/or that are cytotoxic. Active
compounds also include therapeutically active compounds that can be
used in combination with ritanserin or derivatives or analogs
thereof to treat a disease or disorder, such as cancer. In
particular, active compounds include other anti-cancer therapeutic
agents known in the art and compounds that, as described herein,
act synergistically with ritanserin or with a derivative or analog
thereof.
As used herein, the term "derivative" refers to a chemical compound
that can be produced from another compound of similar structure in
one or more chemical or biochemical steps, e.g., via the
replacement of a H by an alkyl, aryl, aralkyl, acyl or amino
group.
As used herein, an "analog" of a chemical compound is a compound
that, by way of example, resembles another in structure but is not
necessarily an isomer (e.g., 5-fluorouracil is an analog of
thymine).
The term "treatment" as used herein refers to any treatment that
has a beneficial effect in treatment of a disease or disorder.
Thus, a treatment can include a therapeutically effective and/or
biologically active compound (e.g., an active compound as described
above and/or chemotherapy), a composition comprising one or more
active compounds or other drugs, a therapeutic biomolecule (e.g.,
an antibody, protein, DNA or RNA) administered to treat a disease
or symptom thereof, surgery, radiation treatment, immunotherapy,
alternating electric field therapy, etc.
"Co-administer" can include simultaneous and/or sequential
administration of two or more treatments.
The term "effective amount" can refer to an amount sufficient to
produce a selected effect, such as alleviating symptoms of a
disease or disorder or of inhibiting an enzyme.
The term "therapeutically effective amount" refers to an amount
(e.g., target plasma concentration or unit dosage) of an active
compound or other treatment (e.g., radiation) sufficient to produce
a beneficial effect to a subject being treated with the compound or
other treatment, such as alleviating symptoms of a disease or
disorder. For example, a therapeutically effective amount can be
the amount of the compound that produces a reduction in tumor size,
reduces or prevents cancer cell metastasis, increases subject
survival, and/or the amount that reduces a symptom of the cancer.
Thus, in the treatment of GBM, a therapeutically effective amount
can be the amount necessary to reduce headaches, seizures, aphasia,
cognitive disorders, nausea or other symptoms.
In the context of administering active compounds in the form of a
combination, such as multiple compounds, the effective amount of
each compound, when administered in combination with another
compound(s), can be different from when that compound is
administered alone. Thus, the effective amount of a combination of
compounds can refer collectively to the combination as a whole,
although the actual amounts of each compound can vary.
The term "more effective" means that the selected effect is
alleviated to a greater extent by one treatment relative to the
second treatment to which it is being compared.
The term "inhibit," as used herein, refers to the ability of a
compound or any agent to reduce or impede a described function,
level, activity, synthesis, release, binding, etc., based on the
context in which the term "inhibit" is used. In some embodiments,
"inhibit" refers to the reduction of the catalytic activity of an
enzyme. In some embodiments, inhibition is by at least 10%, 25%,
50%, or at least 75%. The term "inhibit" is used interchangeably
with "reduce" and "block."
As used herein, "treating" a disease or disorder refers to reducing
the number of diseased (e.g., cancer) cells in a subject,
curtailing the spread of the diseased cells in the subject (e.g.,
preventing metastasis of a cancer), reducing the severity and/or
frequency of one or more effects or symptoms of the disease in a
subject, increasing the survival of a subject being treated for the
disease or disorder, and/or providing any other beneficial effect
to the subject. Thus, treatment is typically administered to a
subject who exhibits signs of pathology for the purpose of
diminishing or eliminating those signs.
As used herein, the term "synergize" and variations thereof (e.g.,
"synergistic") refer to the ability of one treatment to increase
the biological effect (e.g., the cytotoxic effect) of a second
treatment. Synergistic activity, thus, includes but is not limited
to an increased biological effect (e.g., more potent or longer
lasting) using the two treatments (e.g., two drugs) together that
is not observed when the treatments are used separately; a more
effective biological effect, e.g., elimination of multiple symptoms
of a disease; or a reduction in the amount of treatment (e.g., a
reduction in the dosage of a drug) necessary for administration to
achieve the biological effect observed with a single treatment.
Synergistic activity can refer to an interaction or cooperation
between at least two treatments (e.g., two therapeutic compounds or
a therapeutic compound and another therapeutic treatment, such as
radiotherapy or surgery) that enhance or magnifies one or more
biological effect. Thus, in some embodiments, the synergistic
activity produces a combined biological effect greater than the sum
of their separate biological effects. In some embodiments, synergy
refers to a combination of two or more treatments (e.g., two or
more individual active compounds) that has a combination index (CI)
that is less than 1.
The terms "biological sample" or "sample" refer to a sample from a
subject, including, but not limited to, normal tissue samples,
disease tissue samples, biopsies, blood, saliva, feces, semen,
tears, and urine. A sample can also be any other source of material
obtained from a subject which contains cells, cell components,
tissues, and/or a fluid of interest.
The term "cancer" as used herein refers to diseases caused by
uncontrolled cell division and the ability of cells to metastasize,
or to establish new growth in additional sites. The terms
"malignant," "malignancy," "neoplasm," "tumor," "cancer," and
variations thereof refer to cancerous cells or groups of cancerous
cells.
Particular types of cancer include, but are not limited to, skin
cancers (e.g., melanomas), connective tissue cancers (e.g.,
sarcomas), adipose cancers, breast cancers, head and neck cancers,
lung cancers (e.g., mesothelioma), stomach cancers, pancreatic
cancers, ovarian cancers, cervical cancers, uterine cancers,
anogenital cancers (e.g., testicular cancer), kidney cancers,
bladder cancers, colon cancers, prostate cancers, central nervous
system (CNS) cancers (e.g., glioblastomas and gliomas), retinal
cancer, blood, neuroblastomas, multiple myeloma, and lymphoid
cancers (e.g., Hodgkin's and non-Hodgkin's lymphomas).
The terms "anticancer drug," "chemotherapeutic," and "anticancer
prodrug" refer to drugs (i.e., chemical compounds) or prodrugs
known to, or suspected of being able to treat a cancer (i.e., to
kill cancer cells, prohibit proliferation of cancer cells, or treat
a symptom related to cancer). In some embodiments, the term
"chemotherapeutic" as used herein refers to a natural or
non-natural (i.e., synthetic) small molecule that is used to treat
cancer and/or that has cytotoxic ability. Such more traditional or
conventional chemotherapeutic agents can be described by mechanism
of action or by chemical compound class, and can include, but are
not limited to, alkylating agents (e.g., melphalan, temozolomide),
anthracyclines (e.g., doxorubicin), cytoskeletal disruptors (e.g.,
paclitaxel), epothilones, histone deacetylase inhibitors (e.g.,
vorinostat), inhibitors of topoisomerase I or II (e.g., irinotecan
or etoposide), kinase inhibitors (e.g., bortezomib, imatinib,
etc.), nucleotide analogs or precursors thereof (e.g.,
methotrexate), peptide antibiotics (e.g., bleomycin), platinum
based agents (e.g., cisplatin or oxaliplatin), retinoids (e.g.,
tretinoin), and vinka alkaloids (e.g., vinblastine). In some
embodiments, the chemotherapeutic agent is an antibody (e.g., a
human, chimeric, or humanized antibody) or fragment thereof.
The term "cytotoxic" as used herein refers to the ability of a
compound or agent to be toxic to cells, particularly cancer cells.
Cytotoxicity can relate to causing an increase in cell necrosis,
the ability to cause cells to stop growing and/or dividing, and/or
the ability to induce apoptosis in cells. Cytotoxicity can be
observed and/or measured by a variety of assays known in the art,
including, but not limited to, colorometric assays for measuring
cell metabolic activity, e.g., using
3-(4,5-dimethylthiaxol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),
water-soluble tetrazolium (WST) salts, MTS, XTT, or the like;
sulforhodamine B assays; clonogenic assays; ATP-based assays (e.g.,
luciferase assays); and electric cell-substrate impedance sensing
(ECIS).
The term "immunotherapeutic agent" as used herein refers to an
agent that modulates (i.e., activates or suppresses) the immune
response. In some embodiments, the immunotherapeutic agent
stimulates the immune response. In some embodiments, the
immunotherapeutic agent is an immunosuppressant. Immunotherapeutic
agents, include, for example, immunomodulators, such as
interleukins, cytokines (e.g. interferons), chemokines, and
immunomodulatory imide drugs.
The term "pharmaceutical composition" as used herein refers to a
composition comprising at least one active compound, whereby the
composition is amenable for administration to a subject, e.g., to
provide an efficacious outcome in the subject (e.g., a human or
other mammal). Those of ordinary skill in the art will understand
and appreciate the techniques appropriate for determining whether
an active ingredient has a desired efficacious outcome based upon
the needs of the artisan.
As used herein, the term "pharmaceutically acceptable carrier"
includes, by way of example and not limitation, any of the standard
pharmaceutical carriers, such as a phosphate buffered saline
solution, water, emulsions such as an oil/water or water/oil
emulsion, and various types of wetting agents. The term also
encompasses any of the agents approved by a regulatory agency of
the US Federal government or listed in the US Pharmacopeia for use
in animals, including humans.
As used herein, the term "physiologically acceptable" ester or salt
means an ester or salt form of the active ingredient which is
compatible with any other ingredients of the pharmaceutical
composition, and which is not deleterious to the subject to which
the composition is to be administered.
The term "concurrently" when used in the context of the
administration of two or more different active compounds and/or
other treatments can refer to the simultaneous administration of
the two or more active compounds or other treatments (e.g., the
administration of a single composition comprising two or more
active compounds, the administration of multiple compositions, each
comprising an active compound, at roughly the same time (e.g.,
within about 24 hours or less, about 12 hours or less, about 6
hours or less, about 4, hours or less, about 2 hours or less, or
about 30 minutes or less), the administration of a composition
comprising one or more active compounds while a subject is
undergoing a surgery, etc. However, "concurrently" can also be used
to refer to the administration of two different active compounds
and/or other treatments at different times, but while at least one
of the treatments is ongoing (e.g., wherein the subject is being
treated with a course of multiple doses of one active compound or
with radiation over multiple days or weeks and the administration
of a second active compound or other treatment is performed at some
time during the course of the first treatment regimen.
Thus, as used herein, "prior to" in the context of the
administration of multiple active compounds or other treatments can
refer to a situation where a subject has already received a full
course of one active agent or completed another treatment, e.g., a
surgery, before a second treatment is initiated.
As used herein the term "alkyl" refers to C.sub.1-20 inclusive,
linear (i.e., "straight-chain"), branched, or cyclic, saturated or
at least partially and in some cases fully unsaturated (i.e.,
alkenyl and alkynyl) hydrocarbon chains, including for example,
methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl,
pentyl, hexyl, octyl, ethenyl, propenyl, butenyl, pentenyl,
hexenyl, octenyl, butadienyl, propynyl, butynyl, pentynyl, hexynyl,
heptynyl, and allenyl groups. "Branched" refers to an alkyl group
in which a lower alkyl group, such as methyl, ethyl or propyl, is
attached to a linear alkyl chain. In some embodiments, the alkyl
group is "lower alkyl." "Lower alkyl" refers to an alkyl group
having 1 to about 8 carbon atoms (i.e., a C.sub.1-8 alkyl), e.g.,
1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. In some embodiments, the
alkyl is "higher alkyl." "Higher alkyl" refers to an alkyl group
having about 10 to about 20 carbon atoms, e.g., 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, or 20 carbon atoms. In certain embodiments,
"alkyl" refers, in particular, to C.sub.1-8 straight-chain alkyls.
In other embodiments, "alkyl" refers, in particular, to C.sub.1-8
branched-chain alkyls.
Alkyl groups can optionally be substituted (a "substituted alkyl")
with one or more alkyl group substituents, which can be the same or
different. The term "alkyl group substituent" includes but is not
limited to alkyl, substituted alkyl, halo, arylamino, acyl,
hydroxyl, aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl,
aralkylthio, carboxyl, alkoxycarbonyl, oxo, and cycloalkyl. There
can be optionally inserted along the alkyl chain one or more
oxygen, sulfur or substituted or unsubstituted nitrogen atoms,
wherein the nitrogen substituent is hydrogen, lower alkyl (also
referred to herein as "alkylaminoalkyl"), or aryl.
Thus, as used herein, the term "substituted alkyl" includes alkyl
groups, as defined herein, in which one or more atoms or functional
groups of the alkyl group are replaced with another atom or
functional group, including for example, alkyl, substituted alkyl,
halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino,
alkylamino, dialkylamino, sulfate, and mercapto.
The term "aryl" is used herein to refer to an aromatic moiety that
can be a single aromatic ring, or multiple aromatic rings that are
fused together, linked covalently, or linked to a common group,
such as, but not limited to, a methylene or ethylene moiety. The
common linking group also can be a carbonyl, as in benzophenone, or
oxygen, as in diphenylether, or nitrogen, as in diphenylamine. The
term "aryl" specifically encompasses heterocyclic aromatic
compounds. The aromatic ring(s) can comprise phenyl, naphthyl,
biphenyl, diphenylether, diphenylamine and benzophenone, among
others. In particular embodiments, the term "aryl" means a cyclic
aromatic comprising about 5 to about 10 carbon atoms, e.g., 5, 6,
7, 8, 9, or 10 carbon atoms, and including 5- and 6-membered
hydrocarbon and heterocyclic aromatic rings.
The aryl group can be optionally substituted (a "substituted aryl")
with one or more aryl group substituents, which can be the same or
different, wherein "aryl group substituent" includes alkyl,
substituted alkyl, aryl, substituted aryl, aralkyl, hydroxyl,
alkoxyl, aryloxyl, aralkyloxyl, carboxyl, carbonyl, acyl, halo,
nitro, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acyloxyl,
acylamino, aroylamino, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl,
arylthio, alkylthio, alkylene, and --NR'R'', wherein R' and R'' can
each be independently hydrogen, alkyl, substituted alkyl, aryl,
substituted aryl, and aralkyl.
Thus, as used herein, the term "substituted aryl" includes aryl
groups, as defined herein, in which one or more atoms or functional
groups of the aryl group are replaced with another atom or
functional group, including for example, alkyl, substituted alkyl,
halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino,
alkylamino, dialkylamino, sulfate, and mercapto.
Specific examples of aryl groups include, but are not limited to,
cyclopentadienyl, phenyl, furan, thiophene, pyrrole, pyran,
pyridine, imidazole, benzimidazole, isothiazole, isoxazole,
pyrazole, pyrazine, triazine, pyrimidine, quinoline, isoquinoline,
indole, carbazole, and the like.
The term "heteroaryl" refers to aryl groups wherein at least one
atom of the backbone of the aromatic ring or rings is an atom other
than carbon. Thus, heteroaryl groups have one or more non-carbon
atoms selected from the group including, but not limited to,
nitrogen, oxygen, and sulfur.
As used herein, the term "acyl" refers to an organic carboxylic
acid group wherein the --OH of the carboxyl group has been replaced
with another substituent (i.e., as represented by RCO--, wherein R
is an alkyl or an aryl group as defined herein). As such, the term
"acyl" specifically includes arylacyl groups, such as an
acetylfuran and a phenacyl group. Specific examples of acyl groups
include acetyl and benzoyl.
"Cyclic" and "cycloalkyl" refer to a non-aromatic mono- or
multicyclic ring system of about 3 to about 10 carbon atoms, e.g.,
3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. The cycloalkyl group can
be optionally partially unsaturated. The cycloalkyl group also can
be optionally substituted with an alkyl group substituent as
defined herein, oxo, and/or alkylene. There can be optionally
inserted along the cyclic alkyl chain one or more oxygen, sulfur or
substituted or unsubstituted nitrogen atoms, wherein the nitrogen
substituent is hydrogen, alkyl, substituted alkyl, aryl, or
substituted aryl, thus providing a heterocyclic group.
Representative monocyclic cycloalkyl rings include cyclopentyl,
cyclohexyl, and cycloheptyl. Multicyclic cycloalkyl rings include
adamantyl, octahydronaphthyl, decalin, camphor, camphane, and
noradamantyl.
The terms "heterocycle" or "heterocyclic" refer to cycloalkyl
groups (i.e., non-aromatic, cyclic groups as described hereinabove)
wherein one or more of the backbone carbon atoms of a cyclic ring
is replaced by a heteroatom (e.g., nitrogen, sulfur, or oxygen).
Examples of heterocycles include, but are not limited to,
tetrahydrofuran, tetrahydropyran, morpholine, dioxane, piperidine,
piperazine, and pyrrolidine.
"Alkylene" refers to a straight or branched bivalent aliphatic
hydrocarbon group having from 1 to about 20 carbon atoms, e.g., 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or
20 carbon atoms. The alkylene group can be straight, branched or
cyclic. The alkylene group also can be optionally unsaturated
and/or substituted with one or more "alkyl group substituents."
There can be optionally inserted along the alkylene group one or
more oxygen, sulfur or substituted or unsubstituted nitrogen atoms
(also referred to herein as "alkylaminoalkyl"), wherein the
nitrogen substituent is alkyl as previously described. Exemplary
alkylene groups include methylene (--CH.sub.2--); ethylene
(--CH.sub.2--CH.sub.2--); propylene (--(CH.sub.2).sub.3--);
cyclohexylene (--C.sub.6H.sub.10--); --CH.dbd.CH--CH.dbd.CH--;
--CH.dbd.CH--CH.sub.2--;
--(CH.sub.2).sub.q--N(R)--(CH.sub.2).sub.r--, wherein each of q and
r is independently an integer from 0 to about 20, e.g., 0, 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20,
and R is hydrogen or lower alkyl; methylenedioxyl
(--O--CH.sub.2--O--); and ethylenedioxyl
(--O--(CH.sub.2).sub.2--O--). An alkylene group can have about 2 to
about 3 carbon atoms and can further have 6-20 carbons.
"Alkoxyl" or "alkoxy" refers to an alkyl-O-- group wherein alkyl is
as previously described. The term "alkoxyl" as used herein can
refer to, for example, methoxyl, ethoxyl, propoxyl, isopropoxyl,
butoxyl, t-butoxyl, and pentoxyl. The term "oxyalkyl" can be used
interchangably with "alkoxyl".
"Aralkyl" refers to an aryl-alkyl-group wherein aryl and alkyl are
as previously described, and included substituted aryl and
substituted alkyl. Exemplary aralkyl groups include benzyl,
phenylethyl, and naphthylmethyl.
The term "amino" refers to the --NR'R'' group, wherein R' and R''
are each independently selected from the group including H and
substituted and unsubstituted alkyl, cycloalkyl, heterocycle,
aralkyl, aryl, and heteroaryl. In some embodiments, the amino group
is --NH.sub.2.
The term "carbonyl" refers to the --(C.dbd.O)-- or a double bonded
oxygen substituent attached to a carbon atom of a previously named
parent group.
The term "carboxyl" refers to the --COOH group.
The terms "halo", "halide", or "halogen" as used herein refer to
fluoro, chloro, bromo, and iodo groups.
The terms "hydroxyl" and "hydroxy" refer to the --OH group.
The term "oxo" refers to a compound described previously herein
wherein a carbon atom is replaced by an oxygen atom.
The term "cyano" refers to the --CN group.
The term "nitro" refers to the --NO.sub.2 group.
II. General Considerations
Glioblastoma (also referred to as glioblastoma multiforme (GBM)) is
a common cancer originating in the brain. It tends to present with
symptoms such as headache, seizures, aphasia (difficulty with
speech), cognitive deficits, personality changes, and occasionally
nausea/vomiting.
A number of factors render glioblastoma one of the most aggressive
and treatment-resistant cancers. It is highly infiltrative
throughout the brain, making it hard to surgically remove safely
and completely due to the critical nature of many brain structures.
It is also relatively resistant to standard therapies, such as
radiation and chemotherapy. Further, many agents cannot reach
glioblastoma cells adequately due to the blood-brain barrier. A
small sub-population of stem cell-like cells identified within
glioblastoma can also be a component to this treatment
resistance.
Existing therapies provide modest improvements in overall patient
survival. Standard therapy begins with maximal safe surgical
resection of the tumor, followed three to four weeks later by the
initiation of a six-week course of weekday radiation treatments and
daily temozolomide (TMZ) chemotherapy dosing. This is followed by
pulsed TMZ treatment (5 days on, 23 days off), until magnetic
resonance imaging (MRI) or clinical deterioration indicates
progression of the cancer. A 2005 report indicated that the
addition of this TMZ regimen to radiation treatment increase median
survival of patients with glioblastoma from about 12 months to 14.6
months. It also increased two-year survival from 10.4% to 26.4%.
Other therapies, frequently used at times of glioblastoma
progression, include bevacizumab and chemotherapies such as
lomustine and carboplatin. However, none of these have been proven
to improve overall survival.
Glioblastoma, like most cancers, is driven by a diverse set of
genetic (and possibly epigenetic) lesions. Unfortunately, single
targeted therapies have thus far proven ineffective for patients
with the genetically complex and adaptable glioblastoma. One
potential answer lies in treatment with a cocktail of inhibitors.
However, such treatment can lead to increases in toxicity well
ahead of increases in efficacy. A potentially superior alternative
lies in targeting signaling hubs that in one stroke provide the
crippling of multiple critical pathways in glioblastoma. For
instance, the inhibition of diacylglycerol kinase alpha
(DGK.alpha.), a lipid kinase that converts diacylglycerol to
phosphatidic acid, has been shown to act as a signaling hub and a
promising new target in the treatment of glioblastoma. See
Dominguez et al. 2013. Two small-molecule inhibitors of DGK.alpha.,
R59949 and R59022, have been shown to have activity against
glioblastoma and other cancers in vitro and in vivo. See Dominguez
et al. 2013.
The presently disclosed subject matter is based in part on the
finding that ritanserin can also inhibit DGK.alpha.. Ritanserin has
the molecular formula: C.sub.27H.sub.25F.sub.2N.sub.3OS. The IUPAC
name for ritanserin is:
6-[2-[4-[bis(4-fluorophenyl)methylidene]piperidin-1-yl]ethyl]-7-methy-
l-[1,3]thiazolo[3,2-a]py-rimidin-5-one. Thus, ritanserin can be
classified structurally as a derivative of a fused pyrimidine, more
particularly of a thiazolopyrimidine. The chemical structure of
ritanserin includes a 5H-[1,3]thiazolo[3,2-a]pyrimidin-5-one core
that is substituted at position 7 by a methyl group and at position
6 by a 2-{4-[bis(4-fluorophenyl)methylidene]piperidin-1-yl}ethyl
group.
As a potent and long-acting serotonin (5-hydroxytryptamine, 5-HT)
antagonist of the subtype 5-HT2 (Ki=0.39 nM), ritanserin has had
substantial clinical trial experience. It has been used in the
treatment of a variety of disorders including anxiety, depression
and schizophrenia. For instance, it has been shown to be safe and
effective in human trials for the treatment of schizophrenia and
other disorders, such as alcohol addiction, and as a sleep aid. It
has little sedative action. In previous studies for use as a
serotonin receptor inhibitor, ritanserin has also been shown to be
orally bioavailable, to have a 40 hour half-life (see Timmerman et
al., 1989), and to reach the brain in meaningful concentrations.
See Leysen et al., 1985.
The structure of ritanserin is shown in FIG. 1A, alongside those of
R59949 and R59022, for comparison. The structure of ritanserin
differs from that of R59022 by the inclusion of a second aryl
fluoride substituent. As described further hereinbelow, the
presently disclosed subject matter provides data showing that
ritanserin has the ability to inhibit the enzymatic activity of
purified DGK.alpha. and is a more potent inhibitor than the
established DGK.alpha. inhibitor R59022. See FIGS. 1B-1D. Thus,
ritanserin offers the possibility of accelerating the application
of DGK.alpha. inhibition in the clinic.
III. Methods of Inhibiting DGK.alpha. and Treating Cancer
The presently disclosed subject matter is based in part on the
finding that ritanserin can inhibit DGK.alpha. and has activity
against cancer in vitro and in vivo. As described further
hereinbelow, ritanserin has greater activity than the previously
known DGK.alpha. inhibitor R59022. As also described further below,
ritanserin exhibits additional unexpected properties, including the
ability to increase the chemosensitivity and radiosensitivity of
cancer cells, to synergize with other drugs, and to enhance the
synergy of drugs known to synergize with one another.
For example, as described in the Examples below, it is disclosed
herein that ritanserin inhibits DGK.alpha. activity in biochemical
assays. Like R59022, it is strongly cytotoxic against glioblastoma
and melanoma in vitro. In addition, ritanserin toxicity in GBM
cells is largely rescued by administering the DGK.alpha. product
phosphatidic acid (PA), indicating ritanserin action through
DGK.alpha. inhibition. Further, ritanserin can be preferentially
cytotoxic against mesenchymal GBM cells, a particularly
treatment-resistant subset of GBM. Ritanserin increases survival
and decreases tumor volume in mouse models of glioblastoma and
melanoma. The presently disclosed subject matter also identifies
new protein targets of DGK.alpha. inhibition and shows that
ritanserin synergizes with other drugs, such as temozolomide and
chloroquine, in vitro.
Accordingly, in some embodiments, the presently disclosed subject
matter provides compositions and methods for treating cancer,
particularly glioblastoma and melanoma. In some embodiments, the
presently disclosed subject matter provides compositions and
methods to treat glioblastoma, e.g., by regulating the DGK.alpha.
pathway, a cancer therapeutic target. In some embodiments, the
presently disclosed subject matter provides a compound,
composition, or method for inhibiting DGK.alpha..
In some embodiments, a composition comprising a therapeutically
effective amount of ritanserin is administered to a subject in need
thereof. Ritanserin can be understood to be the compound of the
structure displayed at the bottom of FIG. 1A. In some embodiments,
a therapeutically effective amount of a ritanserin analog can be
used in place of the ritanserin in the presently disclosed methods
and compositions. In some embodiments, the ritanserin analog can be
a molecule of the structure shown at the bottom of FIG. 1A, but
including one or more modifications selected from: replacement of
one or more of the fluoride (F) atoms with another electronegative
group, changes in the length and saturation of the ethylene linker
between the piperidine group and the pyrimidinone, the addition of
a substituent to the piperidine group, exchange of the pyrimidinone
group with another heterocycle, replacement of the methyl
substituent with another group, and replacement of the carbonyl
with another group, e.g., C.dbd.NH, etc. Thus, in some embodiments,
a composition comprising a therapeutically effective amount of
ritanserin or a ritanserin analog is administered to the subject,
wherein the ritanserin or ritanserin analog is a compound of the
formula:
##STR00001## wherein:
each of R.sub.1 and R.sub.2 is independently selected from the
group consisting of alkyl, substituted alkyl, --CF.sub.3, alkoxy,
hydroxyl, amino, and halo;
R.sub.3 is hydrogen, hydroxyl, or alkoxy;
R.sub.4 is hydrogen or alkyl;
X.sub.1 is N or CH;
X.sub.2 is O, CH.sub.2, or NH;
X.sub.3 is --S--, --CH.sub.2--, or --CR.sub.5.dbd.CR.sub.6--;
L.sub.1 is alkylene, optionally C.sub.1-C.sub.5 alkylene; and
L.sub.2 is --CH.sub.2CH.sub.2--, CH.sub.2CH.sub.2CH.sub.2--, or
CR.sub.7.dbd.CR.sub.8--, wherein each R.sub.5, R.sub.6, R.sub.7,
and R.sub.8 is independently H, halo, amino, alkyl or substituted
alkyl.
In some embodiments, the subject is a subject who has been
diagnosed with a cancer. In some embodiments, the cancer is
glioblastoma, melanoma, lung cancer, or pancreatic cancer. In some
embodiments, the cancer is a glioblastoma or a melanoma. In some
embodiments, the subject has been newly diagnosed with the cancer
(i.e., the cancer is the first occurrence of the cancer). In some
embodiments, the cancer is a reoccurrence of a previously diagnosed
cancer. In some embodiments, the subject is a mammal. In some
embodiments, the subject is a human. In some embodiments, the
ritanserin is administered orally.
In some embodiments, ritanserin or a ritanserin analog is
administered in combination with at least one additional compound
or treatment (i.e., with at least one "second treatment."). The
additional compound can be a compound known in the art for the
treatment of cancer (e.g., in the treatment of glioblastoma or
melanoma) or a compound known in the art for the treatment of
another disease or disorder. In some embodiments, the additional
compound can be an immunotherapeutic agent. In some embodiments,
the additional compound is selected from temozolomide (TMZ) or
derivatives thereof, e.g., the perillyl alcohol conjugate of TMZ
(TMZ-POH, also known as NEO212), chloroquine, imatinib, or
bevacizumab (tradename Avastin). In some embodiments, the
additional compounds are temozolomide and chloroquine. In some
embodiments, the additional treatment is radiation treatment,
surgery, or alternating electric field therapy. The additional
compound or treatment can be administered at the same time as the
composition comprising ritanserin or a ritanserin analog (i.e.,
concurrently) and/or at another time (i.e., prior to and/or after
administration of the composition comprising ritanserin or a
ritanserin analog).
In some embodiments, ritanserin or a ritanserin analog inhibits
DGK.alpha. activity. In some embodiments, ritanserin or a
ritanserin analog is cytotoxic to cancer cells. In some
embodiments, treatment with ritanserin or a ritanserin analog
increases chemosensitivity of cancer cells to other cytotoxic
agents. In some embodiments, ritanserin or ritanserin analog
treatment of cells increases radiosensitivity.
In some embodiments, the effects of treatment with ritanserin or a
ritanserin analog and one or more additional compounds or
treatments are synergistic. In some embodiments, ritanserin or a
ritanserin analog synergizes with temozolomide. In some
embodiments, ritanserin or a ritanserin analog synergizes with
chloroquine. In some embodiments, ritanserin or a ritanserin analog
potentiates chloroquine synergy with temozolomide. In some
embodiments, ritanserin or a ritanserin analog synergizes with
imatinib. In some embodiments, ritanserin or a ritanserin analog
synergizes with radiation.
In some embodiments, mesenchymal cells are more sensitive to
ritanserin or a ritanserin analog than are non-mesenchymal cells.
In some embodiments, ritanserin or a ritanserin analog is more
effective against proneural cells converted to a mesenchymal
phenotype.
It is disclosed herein that treatment of cancer with ritanserin or
a ritanserin analog increases median survival time. In some
embodiments, the cancer is a mesenchymal cancer. In some
embodiments, the mesenchymal cancer is a glioblastoma.
It is disclosed herein that ritanserin inhibits or suppresses,
inter alia, DGK.alpha. activity, RhoA, GGTase I, NF-KB, p53, and
HSP60. Ritanserin is therefore useful for treating diseases and
disorders where these activities are associated with the disease or
disorder.
In some embodiments, treatment with ritanserin or a ritanserin
analog inhibits tumor growth. In some embodiments, the combination
of ritanserin or a ritanserin analog and another compound or
treatment inhibits the development of chemoresistance and/or
radioresistance. In some embodiments, the combination of ritanserin
or a ritanserin analog and another compound or treatment inhibits a
shift in phenotype (or subtype) of a cancer cell or cells.
In some embodiments, treatment with ritanserin or a ritanserin
analog is at a dosage ranging from about 1.0 mg/kg body weight to
about 500 mg/kg body weight. In some embodiments, the range is from
about 10 mg/kg/body weight to about 250 mg/kg body weight. In some
embodiments, the range is from about 25 mg/kg body weight to about
150 mg/kg body weight. In some embodiments, the dosage is 50 mg/kg
body weight.
One of ordinary skill in the art will appreciate that the dosage of
ritanserin, a ritanserin analog, or other active compound(s) being
administered will depend on the condition being treated, the
particular compound, and other clinical factors such as age, sex,
weight, and health of the subject being treated, the route of
administration of the compound(s), and the type of composition
being administered (tablet, gel cap, capsule, solution, suspension,
inhaler, aerosol, elixir, lozenge, injection, patch, ointment,
cream, etc.). It is to be understood that the presently disclosed
subject matter has application for both human and veterinary
use.
The presently disclosed subject matter further encompasses
biologically active analogs, homologs, derivatives, and
modifications of the compounds of the presently disclosed subject
matter. Methods for the preparation of such compounds are known in
the art.
III. Compositions and Routes of Administration
The presently disclosed subject matter provides, in some
embodiments, compositions comprising ritanserin or a ritanserin
analog. In some embodiments, such compositions can be provided in
the form of pharmaceutical formulations suitable for administration
to a subject in need of treatment for a disease or disorder.
In some embodiments, the presently disclosed subject matter
provides compositions comprising multiple active compounds (i.e.,
multiple medications). In some embodiments, combinations of
multiple active compounds can be used in a single combined
formulation, or used singly (e.g., provided in multiple separate
formulations), within specific dosing ranges to treat cancer. In
some embodiments, two or more medications are administered. In some
embodiments, three or more medications are administered.
When active compounds of the presently disclosed subject matter are
to be administered at the same time, they can be administered in a
formulation containing more than one compound.
In some embodiments, the presently disclosed subject matter
provides a composition comprising ritanserin or a ritanserin analog
and at least one additional pharmaceutically active compound. In
some embodiments, the additional pharmaceutically active compound
is an anti-cancer agent. Thus, in some embodiments, the presently
disclosed subject matter provides a composition comprising
ritanserin or a ritanserin analog and at least one additional
pharmaceutically active compound (e.g., an anti-cancer agent),
wherein the ritanserin or ritanserin analog is a compound of the
formula:
##STR00002## wherein:
each of R.sub.1 and R.sub.2 is independently selected from the
group consisting of alkyl, substituted alkyl, --CF.sub.3, alkoxy,
hydroxyl, amino, and halo;
R.sub.3 is hydrogen, hydroxyl, or alkoxy;
R.sub.4 is hydrogen or alkyl;
X.sub.1 is N or CH;
X.sub.2 is O, CH.sub.2, or NH;
X.sub.3 is --S--, --CH.sub.2--, or --CR.sub.5.dbd.CR.sub.6--;
L.sub.1 is alkylene, optionally C.sub.1-C.sub.5 alkylene; and
L.sub.2 is --CH.sub.2CH.sub.2--, CH.sub.2CH.sub.2CH.sub.2--, or
CR.sub.7.dbd.CR.sub.8--, wherein each R.sub.5, R.sub.6, R.sub.7,
and R.sub.8 is independently H, halo, amino, alkyl or substituted
alkyl. In some embodiments, the at least one additional anti-cancer
agent is known in the art in the treatment of GBM and/or melanoma.
In some embodiments, the presently disclosed subject matter
provides a composition comprising ritanserin or a ritanserin analog
and a second active compound that provides synergistic effect with
ritanserin or a ritanserin analog in the treatment of cancer. In
some embodiments, the composition comprises ritanserin or a
ritanserin analog and at least one of temozolomide, chloroquine,
bevacizumab, imatinib, and an immunotherapeutic agent. In some
embodiments, the composition comprises ritanserin or a ritanserin
analog, and either temozolomide or chloroquine. In some
embodiments, the composition comprises ritanserin or a ritanserin
analog, temozlomide, and chloroquine.
In some embodiments, the presently disclosed subject matter
provides a composition comprising ritanserin and at least one
additional anti-cancer agent.
In some embodiments, the at least one additional anti-cancer agent
is known in the art in the treatment of GBM and/or melanoma. In
some embodiments, the presently disclosed subject matter provides a
composition comprising ritanserin and a second active compound that
provides synergistic effect with ritanserin in the treatment of
cancer. In some embodiments, the composition comprises ritanserin
and at least one of temozolomide, chloroquine, bevacizumab,
imatinib, and an immunotherapeutic agent. In some embodiments, the
composition comprises ritanserin and either temozolomide or
chloroquine. In some embodiments, the composition comprises
ritanserin, temozlomide, and chloroquine.
In some embodiments, the presently disclosed composition also
comprises a pharmaceutically acceptable carrier and/or excipient.
Pharmaceutically acceptable excipients can be selected from the
group including, but not limited to, granulating agents, binding
agents, lubricating agents, disintegrating agents, sweetening
agents, glidants, anti-adherents, anti-static agents, surfactants,
anti-oxidants, gums, coloring agents, flavouring agents, coating
agents, plasticizers, preservatives, suspending agents, emulsifying
agents, spheronization agents and combinations thereof.
Based on the unexpected discoveries described herein, one of
ordinary skill in the art can appreciate that the compounds
disclosed herein as useful for combination drug therapy and/or as
having synergistic effects can in some instances be used singly
instead of as part of a combination. Additionally, based on the
present disclosure, one of ordinary skill in the art will also
appreciate that the active compounds described herein as useful for
combination drug therapy can in some instances be used in any
combination. Until the present discovery of useful combination
therapies as disclosed herein, one or ordinary skill in the art
would not have had such an appreciation.
In some embodiments, at least one of the active compounds being
administered is administered at least once a day. In some
embodiments, it is administered at least twice a day. In some
embodiments, it is administered up to five times a day. In some
embodiments, it is administered up to five times a week. In some
embodiments, it is administered at least once a week. In some
embodiments, it is administered at least once a month.
In some embodiments, at least two or at least three different
active compounds are administered to the subject. It will be
appreciated by one of ordinary skill in the art that the multiple
active compounds, preferably three or more active compounds, being
administered do not necessarily have to be administered at the same
time or in equal doses. In some embodiments, the active compounds
being administered as part of the drug combination therapy are
separately administered. In some embodiments, a first active
compound is administered before a second or third active compound
is administered. In some embodiments, a first active compound and a
second active compound are administered nearly simultaneously,
while a third active compound is administered at a different time.
In some embodiments, the first active compound is administered
subsequent to administration of a second active compound or third
active compound.
The presently disclosed subject matter further provides
pharmaceutical compositions comprising active compounds as
described herein. The pharmaceutical composition can comprise one
or more active compounds, and biologically active analogs,
homologs, derivatives, modifications, and pharmaceutically
acceptable salts thereof, and a pharmaceutically acceptable
carrier. In some embodiments, the active compounds are administered
as a pharmaceutical composition.
The route of administration can vary depending on the type of
compound being administered. In one aspect, the active compounds
are administered via routes such as oral, topical, rectal,
intramuscular, intramucosal, intranasal, inhalation, ophthalmic,
and intravenous.
The presently disclosed subject matter further provides for
administration of an active compound or compounds as a
controlled-release formulation.
The combinations of drugs can be administered in formulations that
contain all drugs being used, or the drugs can be administered
separately. In some cases, it is anticipated that multiple
doses/times of administration will be useful. Additionally, for
some treatment regimens, at least two active compounds will be
used. In some embodiments, at least three compounds will be
administered. The presently disclosed subject matter further
provides for varying the length of time of treatment.
In some embodiments, the results of treating a subject with a
combination of two or more active compounds are additive compared
with the effects of using any of the compounds alone. This does not
mean that if three or more active compounds are administered that
the results will be additive as to the combination of all drugs,
just two or more. In some embodiments, the effects seen when using
two or more active compounds are greater than when using any of the
compounds alone.
In some embodiments, the results of treating a subject with a
combination of two or more active compounds as described herein are
synergistic compared with the effects of using the compounds alone,
at least with regard to two compounds.
Additional compounds can be used to treat subjects according to the
presently disclosed subject matter. In addition to the combination
treatment of at least two drugs, or at least three drugs, described
above, the presently disclosed subject matter further provides for
the administration of at least one additional active compound or
treatment to treat diseases and disorders. Thus, in some
embodiments, four, five, six, seven or more compounds and
treatments can be used in combination to treat a subject.
The presently disclosed subject matter provides for multiple
methods for delivering the active compounds. The compounds can be
provided, for example, as pharmaceutical compositions in multiple
formats as well, including, but not limited to, tablets, capsules,
pills, lozenges, syrups, ointments, creams, elixirs, suppositories,
suspensions, inhalants, injections (including depot preparations),
and liquids.
In some embodiments, a first active compound and a second active
compound are administered nearly simultaneously. In some
embodiments, a first active compound is administered prior to the
second compound. In some embodiments, the first active compound is
administered subsequent to the second compound. If three or more
active compounds are administered, one of ordinary skill in the art
will appreciate that the three or more active compounds can be
administered simultaneously or in varying order.
In some embodiments disclosed herein, an individual is given a
pharmaceutical composition comprising a combination of two or more
active compounds.
In some of these embodiments, each compound is a separate chemical
entity. However, in some embodiments, the at least two compounds
can be joined together by a chemical linkage, such as a covalent
bond, so that the at least two different active compounds form
separate parts of the same molecule. In some embodiments, the
chemical linkage is selected such that after entry into the body,
the linkage is broken, such as by enzymatic action, acid
hydrolysis, base hydrolysis, or the like, and the two separate
active compounds are then formed.
Data from previous structure-activity relationship (SAR) studies
within the art can be used as a guide to determine which compounds
to use and the optimal position or positions on the molecules to
attach the tether such that potency and selectivity of the active
compounds can remain high. The tether or linker moiety can be
chosen from among those of demonstrated utility for linking
bioactive molecules together. Disclosed herein are representative
compounds that can be attached together in different combinations
to form heterobivalent therapeutic molecules.
Examples of linkers reported in the scientific literature include
methylene (CH.sub.2), linkers (see Hussey et al, 2003; and Tamiz et
al., 2001), oligo ethyleneoxy O(--CH.sub.2CH.sub.2O--).sub.n units
used to link naltrexamine to other opioids, glycine oligomers of
the formula
--NH--(COCH.sub.2NH).sub.nCOCH.sub.2CH.sub.2CO--(NHCH.sub.2CO).sub.nNH--
used to link opioid antagonists and agonists together (see
Portoghese et al., 1982; and Portoghese et al., 1986), hydrophilic
diamines used to link opioid peptides together (see Stepinski et
al., 1991), rigid double stranded DNA spacers (Paar et al., 2002)
and the biodegradable linker poly(L-lactic acid). See Klok et al.,
2002. The attachment of the tether to a compound can result in the
compound achieving a favorable binding orientation. The linker
itself can be biodegradable or cannot be biodegradable. The linker
can take the form of a prodrug and be tunable for optimal release
kinetics of the linked drugs. The linker can be either
conformationally flexible throughout its entire length or else a
segment of the tether can be designed to be conformationally
restricted. See Portoghese et al., 1986.
The routes of administration, dosage amounts, and dosage forms
described herein can be utilized for the administration of active
compounds or pharmaceutically acceptable salts thereof for the
treatment of cancer. Suitable forms of the compounds for use in
biologically active compositions and methods of the presently
disclosed subject matter include its pharmaceutically acceptable
salts, polymorphs, solvates, hydrates, and prodrugs.
Administration of an effective amount of at least two active
compounds, or pharmaceutically acceptable salts thereof, whether
alone or in combination with a further therapeutic agent or
treatment, to a subject can detectably treat cancer in the subject.
In exemplary embodiments, administration of at least two active
compounds, or pharmaceutically acceptable salts thereof, whether
alone or in combination with additional therapeutic agents or
treatments, can yield a reduction in tumor volume by at least about
10%, 20%, 30%, 50% or greater, up to about 75-90%, or about 95% or
greater.
The present compositions can optionally comprise a suitable amount
of a pharmaceutically acceptable vehicle so as to provide the form
for proper administration to the patient.
Included within the scope of the presently disclosed subject matter
are various individual anomers, diastereomers and enantiomers as
well as mixtures thereof. In addition, the active compounds
described herein also include any pharmaceutically acceptable
salts, for example: alkali metal salts, such as sodium and
potassium; ammonium salts; monoalkylammonium salts; dialkylammonium
salts; trialkylammonium salts; tetraalkylammonium salts; and
tromethamine salts. Hydrates and other solvates of the compounds
are included within the scope of the presently disclosed subject
matter.
In some embodiments, a composition of the presently disclosed
subject matter can comprise one compound. In some embodiments, a
composition of the presently disclosed subject matter can comprise
more than one compound. In some embodiments, additional drugs or
compounds useful for treating other disorders can be part of the
composition. In some embodiments, a composition comprising only one
compound can be administered at the same time as another
composition comprising at least one other compound. In some
embodiments, the different compositions can be administered at
different times from one another. When a composition comprises only
one compound, an additional composition comprising at least one
additional compound can also be used.
The pharmaceutical compositions useful for practicing the presently
disclosed subject matter can be, for example, administered to
deliver a dose of between 1 ng/kg/day and 100 mg/kg/day.
Pharmaceutical compositions that are useful in the methods of the
presently disclosed subject matter can be administered, for
example, systemically in oral solid formulations, or as ophthalmic,
suppository, aerosol, topical or other similar formulations. In
addition to the appropriate compounds, such pharmaceutical
compositions can contain pharmaceutically-acceptable carriers and
other ingredients known to enhance and facilitate drug
administration. Other possible formulations, such as nanoparticles,
liposomes, resealed erythrocytes, and immunologically based systems
can also be used to administer an appropriate compound, or an
analog, modification, or derivative thereof according to the
methods of the presently disclosed subject matter.
As used herein, "prodrug" refers to an agent that is converted into
the parent drug in vivo. Prodrugs are often useful because, in some
situations, they can be easier to administer than the parent drug.
They can, for instance, be bioavailable by oral administration
whereas the parent is not. The prodrug can also have improved
solubility in pharmaceutical compositions over the parent drug, or
can demonstrate increased palatability or be easier to formulate.
An example, without limitation, of a prodrug would be a prodrug of
an alcohol or carboxylic acid active parent compound which is
administered as an ester (the "prodrug") to facilitate transmittal
across a cell membrane where water solubility is detrimental to
mobility but which then is metabolically hydrolyzed to the
carboxylic acid or alcohol, i.e., the active entity/parent
compound, once inside the cell where water-solubility is
beneficial. A further example of a prodrug might be a short peptide
(polyaminoacid) bonded to a carboxylic acid group of a parent
compound where the peptide is metabolized to provide the active
moiety.
The presently disclosed subject matter encompasses the preparation
and use of pharmaceutical compositions comprising a compound useful
for treatment of the diseases disclosed herein as an active
ingredient. Such a pharmaceutical composition can consist of the
active ingredient alone, in a form suitable for administration to a
subject, or the pharmaceutical composition can comprise the active
ingredient and one or more pharmaceutically acceptable carriers,
one or more additional ingredients, or some combination of these.
The active ingredient can be present in the pharmaceutical
composition in the form of a physiologically acceptable ester or
salt, such as in combination with a physiologically acceptable
cation or anion, as is well known in the art.
The formulations of the pharmaceutical compositions described
herein can be prepared by any method known or hereafter developed
in the art of pharmacology. In general, such preparatory methods
include the step of bringing the active ingredient into association
with a carrier or one or more other accessory ingredients, and
then, if necessary or desirable, shaping or packaging the product
into a desired single- or multi-dose unit.
Although the descriptions of pharmaceutical compositions provided
herein are principally directed to pharmaceutical compositions
which are suitable for ethical administration to humans, it will be
understood by the skilled artisan that such compositions are
generally suitable for administration to animals of all sorts.
Modification of pharmaceutical compositions suitable for
administration to humans in order to render the compositions
suitable for administration to various animals is well understood,
and the ordinarily skilled veterinary pharmacologist can design and
perform such modification with merely ordinary, if any,
experimentation. Subjects to which administration of the
pharmaceutical compositions of the presently disclosed subject
matter is contemplated include, but are not limited to, humans and
other primates, mammals including commercially relevant mammals or
mammals kept as pets, such as cattle, pigs, horses, sheep, cats,
and dogs, and birds including commercially relevant birds and birds
kept as pets, such as chickens, ducks, geese, parrots, and
turkeys.
One type of administration encompassed by the methods of the
presently disclosed subject matter is parenteral administration,
which includes, but is not limited to, administration of a
pharmaceutical composition by injection of the composition, by
application of the composition through a surgical incision, by
application of the composition through a tissue-penetrating
non-surgical wound, and the like. In particular, parenteral
administration is contemplated to include, but is not limited to,
subcutaneous, intraperitoneal, intramuscular, and intrasternal
injection, and kidney dialytic infusion techniques
Pharmaceutical compositions that are useful in the methods of the
presently disclosed subject matter can be prepared, packaged, or
sold in formulations suitable for oral, rectal, vaginal,
parenteral, topical, pulmonary, intranasal, inhalation, buccal,
ophthalmic, intrathecal or another route of administration. Other
contemplated formulations include projected nanoparticles,
liposomal preparations, resealed erythrocytes containing the active
ingredient, and immunologically-based formulations.
A pharmaceutical composition of the presently disclosed subject
matter can be prepared, packaged, or sold in bulk, as a single unit
dose, or as a plurality of single unit doses. As used herein, a
"unit dose" is a discrete amount of the pharmaceutical composition
comprising a predetermined amount of the active ingredient. The
amount of the active ingredient is generally equal to the dosage of
the active ingredient which would be administered to a subject, or
a convenient fraction of such a dosage such as, for example,
one-half or one-third of such a dosage.
The relative amounts of the active ingredient, the pharmaceutically
acceptable carrier, and any additional ingredients in a
pharmaceutical composition of the presently disclosed subject
matter will vary, depending upon the identity, size, and condition
of the subject treated and further depending upon the route by
which the composition is to be administered. By way of example, the
composition cam comprise between 0.1% and 100% (w/w) active
ingredient.
In addition to the active ingredient, a pharmaceutical composition
of the presently disclosed subject matter can further comprise one
or more additional pharmaceutically active agents. Particularly
contemplated additional agents include anti-cancer agents,
anti-emetics, scavengers (e.g., cyanide and cyanate scavengers),
pain relievers, and immunotherapeutic agents.
Controlled- or sustained-release formulations of a pharmaceutical
composition of the presently disclosed subject matter can be made
using conventional technology.
A formulation of a pharmaceutical composition of the presently
disclosed subject matter suitable for oral administration can be
prepared, packaged, or sold in the form of a discrete solid dose
unit including, but not limited to, a tablet, a hard or soft
capsule, a cachet, a troche, or a lozenge, each containing a
predetermined amount of the active ingredient. Other formulations
suitable for oral administration include, but are not limited to, a
powdered or granular formulation, an aqueous or oily suspension, an
aqueous or oily solution, or an emulsion.
As used herein, an "oily" liquid is one which comprises a
carbon-containing liquid molecule and which exhibits a less polar
character than water.
A tablet comprising the active ingredient can, for example, be made
by compressing or molding the active ingredient, optionally with
one or more additional ingredients. Compressed tablets can be
prepared by compressing, in a suitable device, the active
ingredient in a free-flowing form such as a powder or granular
preparation, optionally mixed with one or more of a binder, a
lubricant, an excipient, a surface active agent, and a dispersing
agent. Molded tablets can be made by molding, in a suitable device,
a mixture of the active ingredient, a pharmaceutically acceptable
carrier, and at least sufficient liquid to moisten the mixture.
Pharmaceutically acceptable excipients used in the manufacture of
tablets include, but are not limited to, inert diluents,
granulating and disintegrating agents, binding agents, and
lubricating agents. Known dispersing agents include, but are not
limited to, potato starch and sodium starch glycolate. Known
surface active agents include, but are not limited to, sodium
lauryl sulphate. Known diluents include, but are not limited to,
calcium carbonate, sodium carbonate, lactose, microcrystalline
cellulose, calcium phosphate, calcium hydrogen phosphate, and
sodium phosphate. Known granulating and disintegrating agents
include, but are not limited to, corn starch and alginic acid.
Known binding agents include, but are not limited to, gelatin,
acacia, pre-gelatinized maize starch, polyvinylpyrrolidone, and
hydroxypropyl methylcellulose. Known lubricating agents include,
but are not limited to, magnesium stearate, stearic acid, silica,
and talc.
Tablets can be non-coated or can be coated using known methods to
achieve delayed disintegration in the gastrointestinal tract of a
subject, thereby providing sustained release and absorption of the
active ingredient. By way of example, a material such as glyceryl
monostearate or glyceryl distearate can be used to coat tablets.
Further by way of example, tablets can be coated using methods
described in U.S. Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to
form osmotically-controlled release tablets. Tablets can further
comprise a sweetening agent, a flavoring agent, a coloring agent, a
preservative, or some combination of these in order to provide
pharmaceutically elegant and palatable preparation.
Hard capsules comprising the active ingredient can be made using a
physiologically degradable composition, such as gelatin. Such hard
capsules comprise the active ingredient, and can further comprise
additional ingredients including, for example, an inert solid
diluent such as calcium carbonate, calcium phosphate, or
kaolin.
Soft gelatin capsules comprising the active ingredient can be made
using a physiologically degradable composition, such as gelatin.
Such soft capsules comprise the active ingredient, which can be
mixed with water or an oil medium such as peanut oil, liquid
paraffin, or olive oil.
Lactulose can also be used as a freely erodible filler and is
useful when the compounds of the presently disclosed subject matter
are prepared in capsule form.
Liquid formulations of a pharmaceutical composition which are
suitable for oral administration can be prepared, packaged, and
sold either in liquid form or in the form of a dry product intended
for reconstitution with water or another suitable vehicle prior to
use.
Liquid suspensions can be prepared using conventional methods to
achieve suspension of the active ingredient in an aqueous or oily
vehicle. Aqueous vehicles include, for example, water and isotonic
saline. Oily vehicles include, for example, almond oil, oily
esters, ethyl alcohol, vegetable oils such as arachis, olive,
sesame, or coconut oil, fractionated vegetable oils, and mineral
oils such as liquid paraffin. Liquid suspensions can further
comprise one or more additional ingredients including, but not
limited to, suspending agents, dispersing or wetting agents,
emulsifying agents, demulcents, preservatives, buffers, salts,
flavorings, coloring agents, and sweetening agents. Oily
suspensions can further comprise a thickening agent. Known
suspending agents include, but are not limited to, sorbitol syrup,
hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone,
gum tragacanth, gum acacia, and cellulose derivatives such as
sodium carboxymethylcellulose, methylcellulose, and
hydroxypropylmethylcellulose. Known dispersing or wetting agents
include, but are not limited to, naturally occurring phosphatides
such as lecithin, condensation products of an alkylene oxide with a
fatty acid, with a long chain aliphatic alcohol, with a partial
ester derived from a fatty acid and a hexitol, or with a partial
ester derived from a fatty acid and a hexitol anhydride (e.g.,
polyoxyethylene stearate, heptadecaethyleneoxycetanol,
polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan
monooleate, respectively). Known emulsifying agents include, but
are not limited to, lecithin and acacia. Known preservatives
include, but are not limited to, methyl, ethyl, or n-propyl para
hydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening
agents include, for example, glycerol, propylene glycol, sorbitol,
sucrose, and saccharin. Known thickening agents for oily
suspensions include, for example, beeswax, hard paraffin, and cetyl
alcohol.
In some embodiments, a preparation in the form of a syrup or elixir
or for administration in the form of drops can comprise active
ingredients together with a sweetener, which is preferably
calorie-free, and which can further include methylparaben or
propylparaben as antiseptics, a flavoring and a suitable color.
Liquid solutions of the active ingredient in aqueous or oily
solvents can be prepared in substantially the same manner as liquid
suspensions, the primary difference being that the active
ingredient is dissolved, rather than suspended in the solvent.
Liquid solutions of the pharmaceutical compositions can comprise
each of the components described with regard to liquid suspensions,
it being understood that suspending agents will not necessarily aid
dissolution of the active ingredient in the solvent. Aqueous
solvents include, for example, water and isotonic saline. Oily
solvents include, for example, almond oil, oily esters, ethyl
alcohol, vegetable oils such as arachis, olive, sesame, or coconut
oil, fractionated vegetable oils, and mineral oils such as liquid
paraffin.
Powdered and granular formulations of a pharmaceutical preparation
can be prepared using known methods. Such formulations can be
administered directly to a subject, used, for example, to form
tablets, to fill capsules, or to prepare an aqueous or oily
suspension or solution by addition of an aqueous or oily vehicle
thereto. Each of these formulations can further comprise one or
more of a dispersing or wetting agent, a suspending agent, and a
preservative. Additional excipients, such as fillers and
sweetening, flavoring, or coloring agents, can also be included in
these formulations.
A pharmaceutical composition of the presently disclosed subject
matter can also be prepared, packaged, or sold in the form of oil
in water emulsion or a water-in-oil emulsion. The oily phase can be
a vegetable oil such as olive or arachis oil, a mineral oil such as
liquid paraffin, or a combination of these. Such compositions can
further comprise one or more emulsifying agents including naturally
occurring gums such as gum acacia or gum tragacanth, naturally
occurring phosphatides such as soybean or lecithin phosphatide,
esters or partial esters derived from combinations of fatty acids
and hexitol anhydrides such as sorbitan monooleate, and
condensation products of such partial esters with ethylene oxide
such as polyoxyethylene sorbitan monooleate. These emulsions can
also contain additional ingredients including, for example,
sweetening or flavoring agents.
A pharmaceutical composition of the presently disclosed subject
matter can be prepared, packaged, or sold in a formulation suitable
for rectal administration. Such a composition can be in the form
of, for example, a suppository, a retention enema preparation, and
a solution for rectal or colonic irrigation.
Suppository formulations can be made by combining the active
ingredient with a non-irritating pharmaceutically acceptable
excipient which is solid at ordinary room temperature (i.e. about
20.degree. C.) and which is liquid at the rectal temperature of the
subject (i.e. about 37.degree. C. in a healthy human). Suitable
pharmaceutically acceptable excipients include, but are not limited
to, cocoa butter, polyethylene glycols, and various glycerides.
Suppository formulations can further comprise various additional
ingredients including, but not limited to, antioxidants and
preservatives.
As used herein, "parenteral administration" of a pharmaceutical
composition includes any route of administration characterized by
physical breaching of a tissue of a subject and administration of
the pharmaceutical composition through the breach in the tissue.
Parenteral administration thus includes, but is not limited to,
administration of a pharmaceutical composition by injection of the
composition, by application of the composition through a surgical
incision, by application of the composition through a
tissue-penetrating non-surgical wound, and the like. In particular,
parenteral administration is contemplated to include, but is not
limited to, subcutaneous, intraperitoneal, intramuscular, and
intrasternal injection, and kidney dialytic infusion
techniques.
Formulations of a pharmaceutical composition suitable for
parenteral administration comprise the active ingredient combined
with a pharmaceutically acceptable carrier, such as sterile water
or sterile isotonic saline. Such formulations can be prepared,
packaged, or sold in a form suitable for bolus administration or
for continuous administration. Injectable formulations can be
prepared, packaged, or sold in unit dosage form, such as in ampules
or in multi-dose containers containing a preservative. Formulations
for parenteral administration include, but are not limited to,
suspensions, solutions, emulsions in oily or aqueous vehicles,
pastes, and implantable sustained-release or biodegradable
formulations. Such formulations can further comprise one or more
additional ingredients including, but not limited to, suspending,
stabilizing, or dispersing agents. In some embodiments of a
formulation for parenteral administration, the active ingredient is
provided in dry (i.e., powder or granular) form for reconstitution
with a suitable vehicle (e.g., sterile pyrogen free water) prior to
parenteral administration of the reconstituted composition.
The pharmaceutical compositions can be prepared, packaged, or sold
in the form of a sterile injectable aqueous or oily suspension or
solution. This suspension or solution can be formulated according
to the known art, and can comprise, in addition to the active
ingredient, additional ingredients such as the dispersing agents,
wetting agents, or suspending agents described herein. Such sterile
injectable formulations can be prepared using a non-toxic
parenterally acceptable diluent or solvent, such as water or
1,3-butane diol, for example. Other acceptable diluents and
solvents include, but are not limited to, Ringer's solution,
isotonic sodium chloride solution, and fixed oils such as synthetic
mono- or di-glycerides. Other parentally-administrable formulations
which are useful include those which comprise the active ingredient
in microcrystalline form, in a liposomal preparation, or as a
component of a biodegradable polymer system. Compositions for
sustained release or implantation can comprise pharmaceutically
acceptable polymeric or hydrophobic materials such as an emulsion,
an ion exchange resin, a sparingly soluble polymer, or a sparingly
soluble salt.
Formulations suitable for topical administration include, but are
not limited to, liquid or semi-liquid preparations such as
liniments, lotions, oil in water or water in oil emulsions such as
creams, ointments or pastes, and solutions or suspensions.
Topically-administrable formulations can, for example, comprise
from about 1% to about 10% (w/w) active ingredient, although the
concentration of the active ingredient can be as high as the
solubility limit of the active ingredient in the solvent.
Formulations for topical administration can further comprise one or
more of the additional ingredients described herein.
A pharmaceutical composition of the presently disclosed subject
matter can be prepared, packaged, or sold in a formulation suitable
for pulmonary administration via the buccal cavity. Such a
formulation can comprise dry particles which comprise the active
ingredient and which have a diameter in the range from about 0.5 to
about 7 nanometers, and preferably from about 1 to about 6
nanometers. Such compositions are conveniently in the form of dry
powders for administration using a device comprising a dry powder
reservoir to which a stream of propellant can be directed to
disperse the powder or using a self-propelling
solvent/powder-dispensing container such as a device comprising the
active ingredient dissolved or suspended in a low-boiling
propellant in a sealed container. Preferably, such powders comprise
particles wherein at least 98% of the particles by weight have a
diameter greater than 0.5 nanometers and at least 95% of the
particles by number have a diameter less than 7 nanometers. More
preferably, at least 95% of the particles by weight have a diameter
greater than 1 nanometer and at least 90% of the particles by
number have a diameter less than 6 nanometers. Dry powder
compositions preferably include a solid fine powder diluent such as
sugar and are conveniently provided in a unit dose form.
Low boiling propellants generally include liquid propellants having
a boiling point of below 65.degree. F. at atmospheric pressure.
Generally, the propellant can constitute about 50% to about 99.9%
(w/w) of the composition, and the active ingredient can constitute
about 0.1% to about 20% (w/w) of the composition. The propellant
can further comprise additional ingredients such as a liquid
non-ionic or solid anionic surfactant or a solid diluent
(preferably having a particle size of the same order as particles
comprising the active ingredient).
Pharmaceutical compositions of the presently disclosed subject
matter formulated for pulmonary delivery can also provide the
active ingredient in the form of droplets of a solution or
suspension. Such formulations can be prepared, packaged, or sold as
aqueous or dilute alcoholic solutions or suspensions, optionally
sterile, comprising the active ingredient, and can conveniently be
administered using any nebulization or atomization device. Such
formulations can further comprise one or more additional
ingredients including, but not limited to, a flavoring agent such
as saccharin sodium, a volatile oil, a buffering agent, a surface
active agent, or a preservative such as methylhydroxybenzoate. The
droplets provided by this route of administration preferably have
an average diameter in the range from about 0.1 to about 200
nanometers.
The formulations described herein as being useful for pulmonary
delivery are also useful for intranasal delivery of a
pharmaceutical composition of the presently disclosed subject
matter.
Another formulation suitable for intranasal administration is a
coarse powder comprising the active ingredient and having an
average particle from about 0.2 to about 500 micrometers. Such a
formulation is administered in the manner in which snuff is taken,
i.e., by rapid inhalation through the nasal passage from a
container of the powder held close to the nares.
Formulations suitable for nasal administration can, for example,
comprise from about as little as about 0.1% (w/w) and as much as
about 100% (w/w) of the active ingredient, and can further comprise
one or more of the additional ingredients described herein.
A pharmaceutical composition of the presently disclosed subject
matter can be prepared, packaged, or sold in a formulation suitable
for buccal administration. Such formulations can, for example, be
in the form of tablets or lozenges made using conventional methods,
and can, for example, comprise about 0.1% to about 20% (w/w) active
ingredient, the balance comprising an orally dissolvable or
degradable composition and, optionally, one or more of the
additional ingredients described herein. Alternately, formulations
suitable for buccal administration can comprise a powder or an
aerosolized or atomized solution or suspension comprising the
active ingredient. Such powdered, aerosolized, or atomized
formulations, when dispersed, preferably have an average particle
or droplet size in the range from about 0.1 to about 200
nanometers, and can further comprise one or more of the additional
ingredients described herein.
A pharmaceutical composition of the presently disclosed subject
matter can be prepared, packaged, or sold in a formulation suitable
for ophthalmic administration. Such formulations can, for example,
be in the form of eye drops including, for example, a 0.1% to 1.0%
(w/w) solution or suspension of the active ingredient in an aqueous
or oily liquid carrier. Such drops can further comprise buffering
agents, salts, or one or more other of the additional ingredients
described herein. Other ophthalmically-administrable formulations
which are useful include those which comprise the active ingredient
in microcrystalline form or in a liposomal preparation.
A pharmaceutical composition of the presently disclosed subject
matter can be prepared, packaged, or sold in a formulation suitable
for intramucosal administration. The presently disclosed subject
matter provides for intramucosal administration of compounds to
allow passage or absorption of the compounds across mucosa. Such
type of administration is useful for absorption orally (gingival,
sublingual, buccal, etc.), rectally, vaginally, pulmonary, nasally,
etc.
In some aspects, sublingual administration has an advantage for
active ingredients which in some cases, when given orally, are
subject to a substantial first pass metabolism and enzymatic
degradation through the liver, resulting in rapid metabolization
and a loss of therapeutic activity related to the activity of the
liver enzymes that convert the molecule into inactive metabolites,
or the activity of which is decreased because of this
bioconversion.
In some cases, a sublingual route of administration is capable of
producing a rapid onset of action due to the considerable
permeability and vascularization of the buccal mucosa. Moreover,
sublingual administration can also allow the administration of
active ingredients which are not normally absorbed at the level of
the stomach mucosa or digestive mucosa after oral administration,
or alternatively which are partially or completely degraded in
acidic medium after ingestion of, for example, a tablet.
Sublingual tablet preparation techniques known from the prior art
are usually prepared by direct compression of a mixture of powders
comprising the active ingredient and excipients for compression,
such as diluents, binders, disintegrating agents and adjuvants. In
an alternative method of preparation, the active ingredient and the
compression excipients can be dry- or wet-granulated beforehand. In
some embodiments, the active ingredient is distributed throughout
the mass of the tablet. WO 00/16750 describes a tablet for
sublingual use that disintegrates rapidly and comprises an ordered
mixture in which the active ingredient is in the form of
microparticles which adhere to the surface of water-soluble
particles that are substantially greater in size, constituting a
support for the active microparticles, the composition also
comprising a mucoadhesive agent. WO 00/57858 describes a tablet for
sublingual use, comprising an active ingredient combined with an
effervescent system intended to promote absorption, and also a
pH-modifier.
The compounds of the presently disclosed subject matter can be
prepared in a formulation or pharmaceutical composition appropriate
for administration that allows or enhances absorption across
mucosa. Mucosal absorption enhancers include, but are not limited
to, a bile salt, fatty acid, surfactant, or alcohol. In specific
embodiments, the permeation enhancer can be sodium cholate, sodium
dodecyl sulphate, sodium deoxycholate, taurodeoxycholate, sodium
glycocholate, dimethylsulfoxide or ethanol. In some embodiments, a
compound of the presently disclosed subject matter can be
formulated with a mucosal penetration enhancer to facilitate
delivery of the compound. The formulation can also be prepared with
pH optimized for solubility, drug stability, and absorption through
mucosa such as nasal mucosa, oral mucosa, vaginal mucosa,
respiratory, and intestinal mucosa.
To further enhance mucosal delivery of pharmaceutical agents within
the presently disclosed subject matter, formulations comprising the
active agent can also contain a hydrophilic low molecular weight
compound as a base or excipient. Such hydrophilic low molecular
weight compounds provide a passage medium through which a
water-soluble active agent, such as a physiologically active
peptide or protein, can diffuse through the base to the body
surface where the active agent is absorbed. The hydrophilic low
molecular weight compound optionally absorbs moisture from the
mucosa or the administration atmosphere and dissolves the
water-soluble active peptide. The molecular weight of the
hydrophilic low molecular weight compound is generally not more
than 10000 and preferably not more than 3000. Exemplary hydrophilic
low molecular weight compounds include polyol compounds, such as
oligo-, di- and monosaccharides such as sucrose, mannitol, lactose,
L-arabinose, D-erythrose, D-ribose, D-xylose, D-mannose,
D-galactose, lactulose, cellobiose, gentibiose, glycerin, and
polyethylene glycol. Other examples of hydrophilic low molecular
weight compounds useful as carriers within the presently disclosed
subject matter include N-methylpyrrolidone, and alcohols (e.g.,
oligovinyl alcohol, ethanol, ethylene glycol, propylene glycol,
etc.). These hydrophilic low molecular weight compounds can be used
alone or in combination with one another or with other active or
inactive components of the intranasal formulation.
When a controlled-release pharmaceutical preparation of the
presently disclosed subject matter further contains a hydrophilic
base, many options are available for inclusion. Hydrophilic
polymers such as a polyethylene glycol and polyvinyl pyrrolidone,
sugar alcohols such as D-sorbitol and xylitol, saccharides such as
sucrose, maltose, lactulose, D-fructose, dextran, and glucose,
surfactants such as polyoxyethylene-hydrogenated castor oil,
polyoxyethylene polyoxypropylene glycol, and polyoxyethylene
sorbitan higher fatty acid esters, salts such as sodium chloride
and magnesium chloride, organic acids such as citric acid and
tartaric acid, amino acids such as glycine, beta-alanine, and
lysine hydrochloride, and aminosaccharides such as meglumine are
given as examples of the hydrophilic base. Polyethylene glycol,
sucrose, and polyvinyl pyrrolidone are preferred and polyethylene
glycol are further preferred. One or a combination of two or more
hydrophilic bases can be used in the presently disclosed subject
matter.
The presently disclosed subject matter contemplates pulmonary,
nasal, or oral administration through an inhaler. In some
embodiments, delivery from an inhaler can be a metered dose.
An inhaler is a device for patient self-administration of at least
one active compound as described herein comprising a spray inhaler
(e.g., a nasal, oral, or pulmonary spray inhaler) containing an
aerosol spray formulation of at least one active compound and a
pharmaceutically acceptable dispersant. In some embodiments, the
device is metered to disperse an amount of the aerosol formulation
by forming a spray that contains a dose of at least one active
compound effective to treat a disease or disorder encompassed by
the presently disclosed subject matter. The dispersant can be a
surfactant, such as, but not limited to, polyoxyethylene fatty acid
esters, polyoxyethylene fatty acid alcohols, and polyoxyethylene
sorbitan fatty acid esters. Phospholipid-based surfactants also can
be used.
In some embodiments, the aerosol formulation is provided as a dry
powder aerosol formulation in which an active compound of the
presently disclosed subject matter is present as a finely divided
powder. The dry powder formulation can further comprise a bulking
agent, such as, but not limited to, lactose, sorbitol, sucrose, and
mannitol.
In some embodiments, the aerosol formulation is a liquid aerosol
formulation further comprising a pharmaceutically acceptable
diluent, such as, but not limited to, sterile water, saline,
buffered saline and dextrose solution.
In some embodiments, the aerosol formulation further comprises at
least one additional active compound as described herein in a
concentration such that the metered amount of the aerosol
formulation dispersed by the device contains a dose of the
additional active compound in a metered amount that is effective to
ameliorate the symptoms of disease or disorder disclosed herein
when used in combination with at least a first or second active
compound.
Compounds of the presently disclosed subject matter can be prepared
in a formulation or pharmaceutical composition appropriate for
nasal administration. In some embodiments, the compounds can be
formulated with a mucosal penetration enhancer to facilitate
delivery of the drug. The formulation can also be prepared with pH
optimized for solubility, drug stability, absorption through nasal
mucosa, and other considerations.
Capsules, blisters, and cartridges for use in an inhaler or
insufflator cam be formulated to contain a powder mix of the
pharmaceutical compositions provided herein; a suitable powder
base, such as lactose or starch; and a performance modifier, such
as 1-leucine, mannitol, or magnesium stearate. The lactose can be
anhydrous or in the form of the monohydrate. Other suitable
excipients include dextran, glucose, maltose, sorbitol, xylitol,
fructose, sucrose, and trehalose. The pharmaceutical compositions
provided herein for inhaled/intranasal administration can further
comprise a suitable flavor, such as menthol and levomenthol, or
sweeteners, such as saccharin or saccharin sodium.
For administration by inhalation, the compounds for use according
to the methods of the presently disclosed subject matter are
conveniently delivered in the form of an aerosol spray presentation
from pressurized packs or a nebulizer, with the use of a suitable
propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol, the dosage unit can be
determined by providing a valve to deliver a metered amount.
Capsules and cartridges of e.g., gelatin for use in an inhaler or
insufflator can be formulated containing a powder mix of the drugs
and a suitable powder base such as lactose or starch.
As used herein, "additional ingredients" include, but are not
limited to, one or more of the following: excipients; surface
active agents; dispersing agents; inert diluents; granulating and
disintegrating agents; binding agents; lubricating agents;
sweetening agents; flavoring agents; coloring agents;
preservatives; physiologically degradable compositions such as
gelatin; aqueous vehicles and solvents; oily vehicles and solvents;
suspending agents; dispersing or wetting agents; emulsifying
agents, demulcents; buffers; salts; thickening agents; fillers;
emulsifying agents; antioxidants; antibiotics; antifungal agents;
stabilizing agents; and pharmaceutically acceptable polymeric or
hydrophobic materials. Other "additional ingredients" which can be
included in the pharmaceutical compositions of the presently
disclosed subject matter are known in the art and described, for
example in Genaro, ed., 1985, Remington's Pharmaceutical Sciences,
Mack Publishing Co., Easton, Pa., which is incorporated herein by
reference.
Typically, dosages of the active compounds of the presently
disclosed subject matter which can be administered to an animal,
preferably a human, range in amount from about 1.0 .mu.g to about
100 g per kilogram of body weight of the animal. The precise dosage
administered will vary depending upon any number of factors,
including but not limited to, the type of animal and type of
disease state being treated, the age of the animal and the route of
administration. Preferably, the dosage of the active compound will
vary from about 1 mg to about 10 g per kilogram of body weight of
the animal. More preferably, the dosage will vary from about 10 mg
to about 1 g per kilogram of body weight of the animal.
The active compounds can be administered to a subject as frequently
as several times daily, or it can be administered less frequently,
such as once a day, once a week, once every two weeks, once a
month, or even less frequently, such as once every several months
or even once a year or less. The frequency of the dose will be
readily apparent to the skilled artisan and will depend upon any
number of factors, such as, but not limited to, the type and
severity of the disease being treated, the type and age of the
animal, etc.
The presently disclosed subject matter also includes a kit
comprising the active compound(s) and an instructional material
that describes administration of the compound(s). In some
embodiments, this kit comprises a (preferably sterile) solvent
suitable for dissolving or suspending the composition or
compositions of the presently disclosed subject matter prior to
administering the composition(s) to the mammal.
As used herein, an "instructional material" includes a publication,
a recording, a diagram, or any other medium of expression that can
be used to communicate the usefulness of the active compounds in
the kit for effecting alleviation of the various diseases or
disorders recited herein. Optionally, or alternately, the
instructional material can describe one or more methods of
alleviating the diseases or disorders. The instructional material
of the kit of the presently disclosed subject matter can, for
example, be affixed to a container that contains an active compound
or compounds or be shipped together with a container that contains
the compound or compounds. Alternatively, the instructional
material can be shipped separately from the container with the
intention that the instructional material and the compound(s) be
used cooperatively by the recipient.
One of ordinary skill in the art can determine the dose and term of
treatment to be used.
When two or more compounds are to be administered, they can be
administered in the same pharmaceutical composition or in separate
pharmaceutical compositions. When administered in separate
pharmaceutical compositions, they can be administered
simultaneously or one can be administered first. The amount of time
between administration of the different compounds can vary and can
be determined by one of ordinary skill in the art. For example, the
two compounds could be administered up to 10 minutes apart, up to
30 minutes apart, up to 1 hour apart, etc. In some embodiments, one
or more of the compounds can be administered more than once. In
some embodiments, a compound is administered at least twice. In
some embodiments, a compound is administered at least five times.
In some embodiments, the method is useful for low dose treatment.
In some embodiments, the method is useful for short-term
treatment.
One of ordinary skill in the art can determine the best route of
administration of a pharmaceutical composition of the presently
disclosed subject matter. For example, administration can be
direct, enteral, or parenteral. Enteral includes, for example, oral
and rectal administration. Parenteral includes, for example,
intravenous administration. In some embodiments, administration is
oral administration.
The presently disclosed subject matter further encompasses the use
of therapeutically active homologs, analogs, and derivatives of the
useful compounds of the presently disclosed subject matter.
The presently disclosed subject matter further provides for the use
of a unit dose.
In some embodiments, at least one active compound of the presently
disclosed subject matter can be administered in conjunction with
another therapeutic agent. Additional therapeutic agents include,
for example, antibiotics, anti-diarrheals, steroids,
anti-inflammatories, other antimicrobials, and inducers of
chemokines. In some embodiments, more than one therapeutic agent
can be administered in conjunction with a therapeutic compound of
the presently disclosed subject matter.
The presently disclosed subject matter further provides kits
comprising active compounds, an applicator, and an instructional
material for the use thereof.
EXAMPLES
The following Examples provide further illustrative embodiments. In
light of the present disclosure and the general level of skill in
the art, those of skill will appreciate that the following EXAMPLES
are intended to be exemplary only and that numerous changes,
modifications, and alterations can be employed without departing
from the scope of the presently disclosed subject matter.
Materials and Methods for the Examples
Purification of DGK.alpha.:
To obtain purified protein, 293T cells were cultured in DMEM
containing 5% fetal calf serum and 1% penicillin/streptomycin
(GIBCO.TM., Thermo Fisher Scientific, Waltham, Mass., United States
of America). The cells were transiently transfected with 40 .mu.g
of pcDNA3-FLAG-DGK.alpha. plasmid/plate. The transfection was done
using lipofectamine 2000 at a 2:1 ratio of DNA:lipofectamine. The
cells were harvested and homogenized in lysis buffer (10 mM
Na.sub.2HPO.sub.4, pH 7.4, 50 mM .beta.-glucopyranose, 50 mM NaF, 1
mM EDTA, 1 mM EGTA, 0.02% Triton X-100, phenylmethylsulfonyl
fluoride, leupeptine, and pepstatin). The cell homogenate was
centrifuged at 16,000.times.g at 4.degree. C. for 10 minutes. The
supernatant was immunoprecipitated with anti-FLAG (M2) beads for 2
hours at 4.degree. C. The beads were packed onto a screening column
and washed five times with the lysis buffer. The protein was eluted
with five successive 200 .mu.l fractions of 0.5 mg/ml of FLAG
peptide (LifeTein, LLC, Somerset, N.J., United States of America).
The elution fractions containing DGK.alpha. were combined and
dialyzed three times against 10 mM Na.sub.2HPO.sub.4 (pH 7.4), and
50 mM .beta.-glucopyranose, 50 mM NaF, 1 mM EDTA, and 1 mM EGTA.
The purified DGKs were quantified with UV absorbance and by
comparing DGK.alpha. bands with bovine serum albumin standards on
Coomassie-blue stained SDS-PAGE gels.
Preparation of Liposomes:
Lipids (phosphatidylcholine (PC), diacylglycerol (DAG), and
phosphatidylserine PS)) were co-dissolved in chloroform, dried in
vacuo to form a thin film and hydrated to a final concentration of
19 mM in 50 mM Tris-HCl, 100 mM NaCl (pH 8). The lipids were
subjected to five freeze-thaw cycles and were extruded through a
100 nm diameter polycarbonate filter 11 times. The diameter of the
liposomes was verified using a DynaPro.RTM. Dynamic Light
Scattering instrument from Wyatt Technologies (Santa Barbara,
Calif., United States of America). The final lipid composition of
the liposomes was 5 mol % DAG, 20 mol % PS, and 75 mol % PC.
Kinase Assays:
The enzyme activity was assayed using a final reaction volume of 50
.mu.l containing: 4.75 mM lipids and kinase buffer (100 mM NaCl, 50
mM Tris-HCl, 5 mM MgCl.sub.2, 1 mM CaCl.sub.2, 1 mM DTT). Reactions
were initiated by the addition of 5 .mu.l of 5 mM
[.gamma.-.sup.32P]ATP and were allowed to proceed for 15 minutes at
25.degree. C., then terminated with 250 .mu.l MeOH 0.1N HCl, and
then 500 .mu.l of CHCl.sub.3. The organic phase was washed twice
with 1 M MgCl.sub.2, and 250 .mu.l of the organic phase was used to
measure the extent of .sup.32P incorporation into PA using an
LS6500 Multi-Purpose Scintillation Counter (Beckman Coulter, Brea,
Calif., United States of America). R59022 and ritanserin were
dissolved to 20 mM in DMSO (0.5% (v/v), serially diluted in kinase
buffer and 0.1% BSA (Sigma, St. Louis, Mo., United States of
America) and added to the reaction mixtures.
Cell Lines:
U251 MG human glioblastoma, VMM39 human melanoma, GL261 mouse
glioma, B16 mouse melanoma, and JWL578 human glioblastoma stem cell
line were gifts from Howard Fine (Cornell University, Ithaca, N.Y.,
United States of America), Daniel Gioeli (University of Virginia,
Charlottesville, Va., United States of America), Michael Olin
(University of Minnesota, Minneapolis, Minn., United States of
America), Vic Engelhard (University of Virginia, Charlottesville,
Va., United States of America), and Jeongwu Lee (Cleveland Clinic,
Cleveland, Ohio, United States of America), respectively. All cell
lines are previously described and were maintained in appropriate
serum-containing or serum-free medium. See Dominguez et al., 2013;
Bullock et al., 2001; Kim et al., 2012; Olin et al., 2010; and
Roller et al., 2012. U251MG, VMM39, GL261 and B16 were tested for
authenticity by ATCC in January 2016, and JWL578 was tested for
human species origin in November 2014 and March 2015 by IDEXX
Laboratories (Westbrook, Me., United States of America).
Pharmacological Reagents:
Ritanserin was obtained from Tocris Bioscience (Bristol, United
Kingdom) or Sigma (St. Louis, Mo., United States of America). For
in vitro experiments, ritanserin was dissolved in DMSO at 10 mM
stock solution and maintained at -70.degree. C. For in vivo
experiments, ritanserin was dissolved in corn oil at 10 mg/ml and
administered via oral gavage. Phosphatidic acid (Avanti Polar
Lipids, Alabaster, Ala., United States of America) was suspended in
2 chloroform: 1 methanol at a stock concentration of 5 mM and
maintained at -70.degree. C. until use. Temozolomide (Merck &
Co., Kenilworth, N.J., United States of America) and chloroquine
(Sigma, St. Louis, Mo., United States of America) were dissolved in
dH20 to 10 mM stock solution.
Cell Viability Assays:
In all cases, cells were collected as previously described (see
Dominguez et al., 2013; and Kefas et al., 2013) two to four days
post-treatment with ritanserin or vehicle (v:v DMSO). For cell
counts, attached cells were collected and counted on a
hemocytometer using TrypanBlue exclusion. For cell death assays,
cells were stained with Annexin V-PE and 7-AAD according to the
manufacturer's protocol (BD Pharmingen, Beckton Dickinson and
Company, Franklin Lakes, N.J., United States of America). Single
dye controls were used for compensation. Positive cell staining was
measured using a FACSCALIBUR.TM. flow cytometer (Beckton Dickinson
and Company, Franklin Lakes, N.J., United States of America) and
analyzed with FlowJo software. To assess cell viability, alamarBlue
cell health indicator (ThermoFisher Scientific, Waltham, Mass.,
United States of America) was added to cells and the fluorescent
product measured after 3 to 4 hours in most cases, or after 24
hours in FIG. 6D (excitation 544 nm; emission 590 nm). Experiments
were repeated several times per cell type.
Immunoblotting and Proteomics:
Immunoblotting was performed as previously described. See Dominguez
et al., 2013; and Kefas et al., 2013. Antibodies were obtained from
Cell Signaling Technology (Danvers, Mass., United States of
America). Cells were lysed 24-72 hours post-treatment in 1.times.
lysis buffer. The Human Phospho-Kinase Array kit (R&D Systems,
Minneapolis, Minn., United States of America) was used to identify
ritanserin targets. Immunoblots were performed with multiple
replicates.
Luciferase Reporter Assays:
mTOR promoter reporter-RLuc or HIF-1.alpha. binding site-FLuc
plasmids were transfected into serum-starved U251 and VMM39 one day
after cell plating at 60-80% confluency using Fugene HD at 3:1
(Promega Corporation, Fitchburg, Wis., United States of America).
Cells were fed several hours post-transfection and ritanserin was
added. Cells were lysed after 36-48 hours and assayed with the
Dual-Luciferase Reporter assay system kit (Promega Corporation,
Fitchburg, Wis., United States of America) and Promega GloMax.RTM.
20/20 luminometer (Promega Corporation, Fitchburg, Wis., United
States of America). Results were double-normalized using control
empty vectors pRL and pGL3.
siRNA Transfection:
Lipofectamine.RTM. RNAiMAX transfection reagent (ThermoFisher
Scientific, Waltham, Mass., United States of America) was utilized
for siRNA transfection according to the producer's instructions
with final siRNA concentration of 10 nanamoles per liter (nmol/L).
DGKA siRNAs were as follows: a) custom DGKA siRNA:
5'GGAUUGACCCUGUUCCUAA-3' (SEQ ID NO: 1); b) Dharmacon SMARTpool
ON-TARGETplus (Dharmacon, Lafayette, Colo., United States of
America). The data using the custom DGKA siRNA was generated with
double transfection of glioblastoma stem cells three days
apart.
Orthotopic Tumor Xenografts, Syngeneic Brain Tumor Mouse Models,
and Subcutaneous Tumors:
Intracranial surgical implantation of tumor was as previously
described. See Kefas et al., 2013. In brief, 400,000 U251 human
glioblastoma or 50,000 GL261 mouse glioma suspended in 4 .mu.L of
serum-free DMEM were injected into anesthetized mice through a burr
hole placed 1 mM caudal and 2 mM lateral to the bregma. Treatment
for 8-9 mice per group began 6-7 days post-tumor implantation via
oral gavage and continued daily. Magnetic resonance images were
taken at 3 weeks post-tumor implantation to assess tumor volume,
and volumes were calculated as previously described. See Kefas et
al., 2013. Mice were euthanized at 20% weight loss or with the
first sign of neurological impairment.
For subcutaneous tumors, 250,000 VMM39 human melanoma cells were
injected subcutaneously into the left flank. After two weeks,
tumors were measured daily using electronic calipers and tumor
volume calculated. Mice were euthanized when tumors were greater
than 15 mM in any dimension or greater than 1000 mm.sup.3 in
volume.
Statistics:
Statistical analyses were performed using GraphPad Prism 6
(GraphPad Software, Inc., La Jolla, Calif., United States of
America) and CompuSyn (ComboSyn, Inc., Paramus, N.J., United States
of America). One-way ANOVA with multiple comparisons to generate
p-values was used to analyze single-drug in vitro experiments of
more than two doses. Two-way ANOVA and Chou-Talalay model
combination index was used to determine statistically likely drug
synergies in combination experiments. For Chou-Talalay, percent
inhibition (0.01 to 0.99) of alamarBlue signal was used to
calculate average dose-responses (Fa) from three or more technical
replicates per treatment group. Chou-Talalay combination indices
(CI) less than 1 were considered to be synergistic, and less than
0.2 were an indication of strong synergy. See Chou, 2010. In vivo
experiments were analyzed with the Kaplan-Meier survival function
to determine p-values. Refutation of the null hypothesis was
accepted for p<0.05.
Example 1
Inhibition of DGK.alpha.
DGK.alpha. activity was tested in vitro against increasing
concentrations of both R59022 and ritanserin using liposomes and
the purified enzyme.
The IC.sub.50 values were 20 .mu.M for R59022 and 9 .mu.M for
ritanserin. See FIGS. 1B and 1C. The IC.sub.50 of R59022 obtained
was similar to that found by others in assays using cellular
homogenates. See Sato et al., 2013. Ritanserin is a known serotonin
receptor inhibitor at very low nanomolar doses, but the presently
disclosed data shows that it can be repurposed as an inhibitor of
DGK.alpha. in the low micromolar range, as well.
Example 2
Ritanserin Cytotoxicity in Glioblastoma and Melanoma Cell Lines
Several cancer cell lines were tested in vitro to determine whether
ritanserin was cytotoxic. A single dose of ritanserin was
sufficient to suppress cell growth in GBM lines U251 (human), GL261
(mouse), and JWL578 (human GBM stem cell line) with IC.sub.50s 5-10
.mu.M, very similar to previous results with R59022. See FIG. 2A.
See also, Dominguez et al., 2013. Melanoma lines VMM39 (human) (see
FIG. 2A) and B16 (mouse) (see FIG. 2E) were also affected, with
IC.sub.50s of 10-15 .mu.M. See FIG. 2A. Data from alamarBlue
assays, a measure of cellular viability, provided similar
IC.sub.50s. Immortalized astrocytes were viable above 20 .mu.M
ritanserin. These low-micromolar IC.sub.50s are comparable to the
concentrations at which ritanserin inhibits DGK.alpha.. See FIG.
1C.
To determine the mechanism of ritanserin cytotoxicity,
ritanserin-treated cells were assayed for Annexin V-positivity as
well as cellular permeability (with DNA dye 7-AAD). Both U251 GBM
and VMM39 melanoma cell lines demonstrated increased Annexin V
after ritanserin treatment compared to controls. See FIG. 2B and
FIG. 2F. However, pre-treatment of cells with apoptosis inhibitor
ZVAD-FMK did not rescue cell viability with ritanserin treatment.
See FIG. 2G. These results indicated that ritanserin is directly
cytotoxic to GBM, but that the mechanism might be inducing cell
death upstream of caspase activation.
Given these data, chloroquine, an anti-autophagic reagent, and
necrostatin-1, an anti-necroptotic agent, were tested in
combination with ritanserin to determine whether either would
rescue cell viability. Interestingly, ritanserin and chloroquine
were synergistically cytotoxic. See FIG. 2C. LC3-II accumulated
with ritanserin treatment or a combination of ritanserin and
chloroquine treatment, suggesting autophagy induction. See FIG. 2C
and FIG. 2H. Chloroquine is being investigated as a therapeutic
adjunct in many cancers. See Kimura et al., 2013. The observed
autophagy induction by ritanserin appears to be protective, given
that chloroquine enhanced cancer cell killing by ritanserin.
Necrostatin-1 did not show any rescue of ritanserin toxicity,
suggesting that necroptosis is not a major mechanism of ritanserin
cell death. See FIG. 2G. In U251 GBM, cleaved Poly-ADP ribose
Polymerase (PARP) was observed in ritanserin-treated cells via
western blot (see FIG. 2D), suggesting some degree of
apoptosis.
Example 3
Rescue with Phosphatidic Acid and Suppression of DGK.alpha.
Targets
Given that ritanserin can inhibit DGK.alpha. in a kinase assay,
rescue of ritanserin cytotoxicity in vitro was attempted with
phosphatidic acid, the immediate downstream product of DGK.alpha.'s
ATP-dependent catalytic addition of a phosphate to diacylglycerol
(DAG). The addition of phosphatidic acid (PA) to the culture medium
substantially rescued ritanserin treatment in U251 and JWL578 GBM
cells. See FIG. 3A and FIG. 3C.
To compare to previous findings that DGK.alpha. inhibition affects
mTOR transcription, U251 and VMM39 were transfected with an mTOR
promoter reporter and treated with ritanserin or vehicle. See FIG.
3B. Ritanserin treatment strongly decreased mTOR promoter activity.
U251 treatment with ritanserin also resulted in a loss of
HIF-1.alpha. reporter activity. See FIG. 3B. Ritanserin therefore
appears to mimic R59022's effects on known DGK.alpha. targets in
the mTOR and HIF-1.alpha. signaling pathways. See Dominguez et al.,
2013.
Example 4
Sensitization to DNA-damaging Agents with Ritanserin
A dot-blot proteomics assay was used to investigate ritanserin
downstream targets. U251 treated with 15 .mu.M ritanserin were
lysed at 24 hours. See FIG. 4A. Several target proteins were
substantially changed with ritanserin treatment. HSP60 decreased to
10% of baseline. Heat shock proteins are molecular targets in
glioblastoma, and their loss prevents the unfolded protein
response. See Ampie et al., 2015. HIF-1a and possibly mTOR
up-regulate HSP60. P70 S6K phosphorylation at T389/T421 was
decreased, as expected given DGK.alpha. activation of mTOR. See
FIG. 4A.
Decreased p53 S15 can cause chemosensitization to other cytotoxic
reagents. p53 is hyper-phosphorylated after serine 15
phosphorylation in DNA-damaged cells as an anti-apoptotic,
pro-transcriptional pathway, and antagonism of S15 phosphorylation
is associated with increasing sensitivity to DNA-damaging agents.
See Loughery et al., 2014; and Wittlinger et al., 2007. Immunoblots
of extracts from both U251 and VMM39 cell lines confirmed that
ritanserin decreases S15 phosphorylation. See FIG. 4B.
Example 5
In Vivo Anti-Cancer Activity
Since ritanserin is known to cross the blood-brain barrier and is
orally bioavailable, glioblastoma survival experiments were
undertaken in both orthotopic xenograft and syngeneic tumor models
to determine the effect of oral ritanserin in vivo. Athymic nu/nu
mice were injected with U251 cells and one week later began daily
oral gavage treatment. 50 mg/kg daily dosing of ritanserin
increased survival significantly. See FIG. 5A.
DGK.alpha. inhibition decreases T-cell anergy. Thus, it is believed
that a syngeneic, immunocompetent tumor model could show a stronger
survival benefit. GL261 mouse glioma cells were implanted
intracranially and treatment begun six days later. Ritanserin
improved median survival to 64 days versus 36 days with vehicle.
See FIG. 5B. Magnetic resonance imaging showed decreased tumor size
compared to vehicle. See FIG. 5B. A subcutaneous melanoma tumor
model delayed tumor formation in 50 mg/kg ritanserin-treated mice
compared to vehicle and 10 mg/kg ritanserin-treated mice. See FIG.
5C. Ritanserin treatment also yielded decreases in tumor size and
prolonged survival in other cell lines and xenograft models. See
FIGS. 5D and 5E.
Example 6
Ritanserin Synergy with Temozolomide and/or Chloroquine
The presently disclosed data related to downstream targets of
ritanserin suggest that ritanserin could synergize with known
cytotoxic chemotherapies. Combination therapy experiments were
performed using ritanserin and temozolomide, the standard-of-care
chemotherapy for glioblastoma. Notably, ritanserin and temozolomide
strongly synergized against U251 and VMM39 using alamarBlue assay.
See FIGS. 6A and 6B. Concentrations of ritanserin as low as 7.5
.mu.M with temozolomide at 50 .mu.M decreased cellular viability by
up to 90%, whereas single drug treatment at these concentrations
did not impact cell viability significantly. Chou-Talalay analysis
indicated synergy. See FIG. 6C. Similar results were also
demonstrated in a G2 mesenchymal GBM stem cell line. See FIG. 6E. A
ritanserin, temozolomide, and chloroquine combination experiment
showed strong synergy as well in U251 cells. See FIG. 6D. This
assay was developed over 24 hours to yield a very broad range of
dose-effects (Fa). Temozolomide and chloroquine have previously
been found to synergize against glioblastoma. See Hori et al.,
2015; and Lee et al., 2015. Such synergy was also observed here at
higher doses of chloroquine (50 .mu.M).
As described in Example 2, ritanserin is also synergistically
cytotoxic when used with chloroquine alone. See FIG. 2C. FIGS.
7A-7H provide additional data showing that ritanserin and
chloroquine have cytotoxic synergy in glioblastoma and melanoma
cells, but not astrocytes. Overall, the present results suggest
ritanserin as a novel DGK.alpha. inhibitor, with promising activity
alone or in combination against glioblastoma and melanoma cells,
and could speed the translation of DGK.alpha. inhibition to the
clinic.
Example 7
Toxicity for Mesenchymal Glioblastoma Subtype
Glioblastomas can be categorized into four different subtypes based
on gene expression patterns and clinical characterization. In vitro
ritanserin treatment of 11 different GBM cell lines of various
sub-types indicated that meschenchymal GBM cell lines are three to
four times more sensitive to ritanserin treatment than other
subtypes. See FIG. 8A. Mesenchymal GBM cells were also more
sensitive to knock down of DGK.alpha. expression with small
interfering RNAs (siRNAs). See FIG. 8E. In vivo studies also showed
that there was increased median survival with ritanserin treatment
in a mesenchymal GBM model. In particular, median survival
increased from 17.5 days to 28.5 days in mice with xenografts from
a G88 mesenchymal GBM stem cell line. See FIG. 8C. Median survival
was also increased with oral ritanserin treatment in mice with
xenografts from a G2 mesenchymal GBM stem cell line. See FIG.
8F.
Ritanserin was also more effective in proneural cell lines
following conversion to a mesenchymal phenotype induced by
TNF-alpha treatment. See FIG. 8B. In addition, using established
epithelial-mesenchymal transition (EMT) models, both lung (A549)
and pancreatic (MPanc-96) carcinoma cells became more sensitive to
ritanserin post-EMP. See FIG. 8D. Thus it appears that the
mesenchymal cancer phenotype can be preferentially targeted with
ritanserin in GBM and in other cancers, as well.
Example 8
Radiosensitization with Ritanserin
Since radiation is a frontline treatment for GBM, the combination
of ritanserin treatment and radiotherapy was tested. The
radiosensitization of two different mesenchymal GBM stem cells,
including a radioresistant cell type (i.e., G88 cells) is shown in
FIGS. 9A and 9B. In particular, while the radio-resistant cell type
showed more colonies post-radiation when radiation was used alone,
ritanserin had a clear radiosensitization effect. See FIG. 9A.
Chou-Talalay analysis indicated synergy. Ritanserin treatment also
appears to affect downstream regulators of the radiation response.
See FIG. 9C. NF-.kappa.B has been indicated as a factor in
radioresistance. Thus, the effect of ritanserin on NF-.kappa.B
expression was also measured in two mesenchymal GBM cell lines
using a NF-.kappa.B luciferase assay. Results showed that
ritanserin treatment decreased NF-.kappa.B expression. See FIGS. 9D
and 9E.
Example 9
Effect on Additional Downstream Mediators
Ritanserin also appears to inhibit RhoA expression in G2 and G88
mesenchymal GBM cells based on RhoA ELISA. See FIG. 10A. Further,
based on increases in unprenylated ras-related protein Rap1A in
various GBM cells, it appears that ritanserin inhibits
geranylgeranyltransferase type 1 (GGTase I). See FIG. 10B.
Example 10
Synergy with Drugs with Anti-Proneural Activity
Recent reports have indicated that shifts between GBM subtypes can
be a mechanism of treatment resistance. See Mao et al., 2013; and
Bhat et al., 2013. Mesenchymal GBM stem cells (GSCs) showed
increased expression of key proneural markers OLIG2 and SOX2,
suggesting potential mesenchymal-proneural transition (MPT) as a
drug resistance mechanism. Activation of the PDGFR.alpha. pathway
is another hallmark of the proneural GBM subtype. See Verhaak et
al., 2010; and Phillips et al., 2006. Greater PDGFR.alpha.
activation was observed with ritanserin treatment. See FIG. 11A.
Combination of ritanserin with imatinib, an inhibitor of
PDGFR.alpha. and other receptor tyrosine kinases found to have
preferential activity against proneural GBM, showed significant
synergy against mesenchymal GSCs in vitro (see FIG. 11B) and in
vivo. CI values for G2 and G88 cells were 0.24 and 0.25,
respectively.
REFERENCES
All references listed in the instant disclosure, including but not
limited to all patents, patent applications and publications
thereof, and scientific journal articles are incorporated herein by
reference in their entireties to the extent that they supplement,
explain, provide a background for, and/or teach methodology,
techniques, and/or compositions employed herein. The discussion of
the references is intended merely to summarize the assertions made
by their authors. No admission is made that any reference (or a
portion of any reference) is relevant prior art. Applicants reserve
the right to challenge the accuracy and pertinence of any cited
reference. Ampie L, Choy W, Lamano J B, Fakurnejad S, Bloch O,
Parsa A T. Heat shock protein vaccines against glioblastoma: from
bench to bedside. J Neurooncol. 2015; 123(3):441-448. Bhat K P,
Balasubramaniyan V., Vaillant B., et al. Mesenchymal
differentiation mediated by NF-kappaB promotes radiation resistance
in glioblastoma. Cancer Cell. 2013; 24(3): 331-346. Bullock T N,
Mullins D W, Colella T A, Engelhard V H. Manipulation of avidity to
improve effectiveness of adoptively transferred CD8(+) T cells for
melanoma immunotherapy in human MHC class 1-transgenic mice. J
Immunol. 2001; 167(10): 5824-5831. de Chaffoy de Courcelles D C,
Roevens P, Van Belle H. R 59 022, a diacylglycerol kinase
inhibitor. Its effect on diacylglycerol and thrombin-induced C
kinase activation in the intact platelet. J Biol Chem. 1985;
260(29):15762-15770. Chou T C. Drug combination studies and their
synergy quantification using the Chou-Talalay method. Cancer Res.
2010; 70(2):440-446. Domingo-Musibay E, Galanis E. What next for
newly diagnosed glioblastoma? Future Oncol. 2015. Dominguez C L,
Floyd D H, Xiao A, et al. Diacylglycerol kinase alpha is a critical
signaling node and novel therapeutic target in glioblastoma and
other cancers. Cancer Discov. 2013; 3(7):782-797. Hori Y S, Hosoda
R, Akiyama Y, et al. Chloroquine potentiates temozolomide
cytotoxicity by inhibiting mitochondrial autophagy in glioma cells.
J Neurooncol. 2015; 122(1):11-20. Hussey et al., J. Am. Chem. Soc.
2003; 125:3692-3693. Kefas B, Floyd D H, Comeau L, et al. A
miR-297/hypoxia/DGK-alpha axis regulating glioblastoma survival.
Neuro Oncol. 2013; 15(12):1652-1663. Kim Y, Kim K H, Lee J, et al.
Wnt activation is implicated in glioblastoma radioresistance. Lab
Invest. 2012; 92(3):466-473. Kimura T, Takabatake Y, Takahashi A,
Isaka Y. Chloroquine in cancer therapy: a double-edged sword of
autophagy. Cancer Res. 2013; 73(1):3-7. Klok et al., Macromolecules
2002; 35:746-759. Lee S W, Kim H K, Lee N H, et al. The synergistic
effect of combination temozolomide and chloroquine treatment is
dependent on autophagy formation and p53 status in glioma cells.
Cancer Lett. 2015; 360(2):195-204. Leysen J E, Gommeren W, Van
Gompel P, Wynants J, Janssen P F, Laduron P M. Receptor-binding
properties in vitro and in vivo of ritanserin: A very potent and
long acting serotonin-S2 antagonist. Mol Pharmacol. 1985;
27(6):600-611. Loughery J, Cox M, Smith L M, Meek D W. Critical
role for p53-serine 15 phosphorylation in stimulating
transactivation at p53-responsive promoters. Nucleic Acids Res.
2014; 42(12):7666-7680. Mao P, Joshi K, Li J, et al. Mesenchymal
glioma stem cells are maintained by activated glycolytic metabolism
involving aldehyde dehydrogenase 1A3. Proc Natl Acad Sci USA. 2013;
110(21): 8644-8649. Nobe K, Ohata H, Momose K. Effect of
diacylglycerol kinase inhibitor, R59022 on cytosolic free calcium
level and force development in guinea pig taenia coli. Res Commun
Chem Pathol Pharmacol. 1993; 81(3):331-343. Olenchock B A, Guo R,
Carpenter J H, et al. Disruption of diacylglycerol metabolism
impairs the induction of T cell anergy. Nat Immunol. 2006;
7(11):1174-1181.\ Olin M R, Andersen B M, Zellmer D M, et al.
Superior efficacy of tumor cell vaccines grown in physiologic
oxygen. Clin Cancer Res. 2010; 16(19):4800-4808. Ostrom Q T,
Gittleman H, Fulop J, et al. CBTRUS Statistical Report: Primary
Brain and Central Nervous System Tumors Diagnosed in the United
States in 2008-2012. Neuro Oncol. 2015; 17 Suppl 4:iv1-iv62. Paar
et al., J. Immunol. 2002; 169:856-864. Phillips H S, Kharbanda S,
Chen R, et al. Molecular subclasses of high-grade glioma predict
prognosis, delineate a pattern of disease progression, and resemble
stages in neurogenesis. Cancer Cell. 2006; 9(3):157-173. Portoghese
et al., Life Sci. 1982; 31:1283-1286. Portoghese et al., J. Med.
Chem. 1986; 29:1855-1861. Roller D G, Axelrod M, Capaldo B J, et
al. Synthetic lethal screening with small-molecule inhibitors
provides a pathway to rational combination therapies for melanoma.
Mol Cancer Ther. 2012; 11(11):2505-2515. Sato M, Liu K, Sasaki S,
et al. Evaluations of the selectivities of the diacylglycerol
kinase inhibitors R59022 and R59949 among diacylglycerol kinase
isozymes using a new non-radioactive assay method. Pharmacology.
2013; 92(1-2):99-107. Stepinski et al., Internat. J. of Peptide
& Protein Res. 1991; 38:588-92. Tamiz et al., J. Med. Chem.
2001; 44:1615-1622. Timmerman P, Woestenborghs R, Lenoir H,
Heykants J. Deuterated ritanserin analysis by gas
chromatography/mass spectrometry: a sensitive technique to study
human ritanserin pharmacokinetics. Biomed Environ Mass Spectrom.
1989; 18(7):498-502. U.S. Pat. No. 4,160,452. U.S. Pat. No.
4,256,108. U.S. Pat. No. 4,265,874. Verhaak R G, Hoadley K A,
Purdom E, et al., Integrated genomic analysis identifies clinically
relevant subtypes of glioblastoma characterized by abnormalities in
PDGFRA, IDH1, EGFR, and NF1. Cancer Cell. 2010; 17(1):98-110.
Wittlinger M, Grabenbauer G G, Sprung C N, Sauer R, Distel L V.
Time and dose-dependent activation of p53 serine 15 phosphorylation
among cell lines with different radiation sensitivity. Int J Radiat
Biol. 2007; 83(4):245-257. WO 00/16750. WO 00/57858. Zha Y, Marks
R, Ho A W, et al. T cell anergy is reversed by active Ras and is
regulated by diacylglycerol kinase-alpha. Nat Immunol. 2006;
7(11):1166-1173.
It will be understood that various details of the presently
disclosed subject matter may be changed without departing from the
scope of the presently disclosed subject matter. Furthermore, the
foregoing description is for the purpose of illustration only, and
not for the purpose of limitation.
SEQUENCE LISTINGS
1
1119RNAArtificial Sequencesynthetic sequence 1ggauugaccc uguuccuaa
19
* * * * *